Structures and stability of N<Subscript>13</Subscript><Superscript>+</Superscript> and N<Subscript>13</Subscript><Superscript>−</Superscript> clusters

The structures and stabilities of eleven N₁₃ ⁺ and N₁₃ ^– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N₁₃ ⁺ isomers and six N₁₃ ^– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N₁₃ ⁺ cation is structure C-2 with C_2v symmetry, which contains a pentazole ring and two N₄ open chains. It is different from those of the N₇ ⁺ and N₉ ⁺ clusters, but similar to the N₁₁ ⁺ cluster. Meanwhile, the most stable N₁₃ ^– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N₇ ^– and N₉ ^– clusters, but also from the N₁₁ ^– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species. Figure Optimized geometrical parameters of N₁₃ ⁺ isomer C-2      

Flow of Deposited Inorganic N in Two Gleysol-dominated Mountain Catchments Traced with <Superscript>15</Superscript>NO<Subscript>3</Subscript><Superscript>−</Superscript> and <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript>

In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of ¹⁵NO₃ and ¹⁵NH₄ were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately 1600 m², were delimited by trenches in the Gleysols. K¹⁵NO₃ was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition. After the sampling and a one-year break, ¹⁵NH₄Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood), ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period, the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO₃⁻ than NH₄⁺ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of ¹⁵NO₃⁻ in runoff, with sharp ¹⁵N peaks corresponding to discharge peaks. NO₃⁻ leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline of its C:N ratio.     

Molecular modeling of the rabbit colonic (HKα2a) H<Superscript>+</Superscript>, K<Superscript>+</Superscript> ATPase

A model of the HK2a subunit of the rabbit colonic H⁺, K⁺ ATPase has been generated using the crystal structure of the Ca⁺² ATPase as a template. A pairwise sequence alignment of the deduced primary sequences of the two proteins demonstrated that they share 29% amino acid sequence identity and 47% similarity. Using O (version 7) the model of HK2a was constructed by interactively mutating, deleting, and inserting the amino acids that differed between the pairwise sequence alignment of the Ca⁺² ATPase and HK2a. Insertions and deletions in the HK2a sequence occur in apparent extra-membraneous loop regions. The HK2a model was energy minimized and globally refined to a level comparable to that of the Ca⁺² ATPase structure using CNS. The charge distribution over the surface of HK2a was evaluated in GRASP and possible secondary structure elements of HK2a were visualized in BOBSCRIPT. Conservation and placement of residues that may be involved in ouabain binding by the H⁺, K⁺ ATPase were considered and a putative location for the subunit was postulated within the structure.Figure Possible architecture of the HK2a subunit. The residue in green is the lysine (position 517, Fig. 1) that lies in the nucleotide binding pocket and the residue in red is the aspartic acid at the phosphorylation site (position 385). Based on an alignment with the Ca⁺² ATPase, ten transmembrane helices were modeled into HK2a. The ten transmembrane helices are drawn as rods and shown in different colors for clarity. From left to right, the transmembrane helix designations are M10 (blue), M7 (gray), M8 (purple), M9 (orange), M5 (pink), M6 (green), M3 (brown), M4 (cyan), M2 (teal), and M1 (almond).     

Different effects of SNP and GSNO on mitochondrial O<Stack><Subscript>2</Subscript><Superscript>.−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H₂O₂ release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ( ^. NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its ^. NO release) induces an inhibition of the succinate dependent H₂O₂ production consistent with a ^. NO dependent covalent modification. However maximal inhibition of the succinate dependent H₂O₂ release is obtained in the presence of low GSNO (20–100 μM), but not with SNP. This inhibition appears independent of ^. NO release since μM GSNO does not affect mitochondrial respiration, or the H₂O₂ detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O₂^.− since GSNO chemically competes with NBT and cytochrome C in O₂^.− detection.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

Rapid measurement of <Superscript>3</Superscript>J(H<Superscript>N</Superscript>–H<Superscript>α</Superscript>) and <Superscript>3</Superscript>J(N–H<Superscript>β</Superscript>) coupling constants in polypeptides

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.     

CD14<Superscript>++</Superscript>CD16<Superscript>−</Superscript> and CD14<Superscript>+</Superscript>CD16<Superscript>+</Superscript> human monocyte adhesion to endothelial cells

Based on the difference in the CD14 and CD16 expression, two subsets of monocytes were identified in human and other mammalian blood. These subsets have different patterns of adhesion molecules and chemokine receptors that suggests the different mode of their interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14⁺CD16⁺ and CD14⁺⁺CD16⁻ monocytes to adhere to endothelial cell monolayer in presence or absence of pro- and anti-inflammatory cytokines. We demonstrated that CD14⁺CD16⁺ monocytes had a higher level of adhesion to intact monolayer of endothelial cells than CD14⁺⁺CD16⁻ monocytes. Adhesion of CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes significantly increased in the presence of TNFα or its combination with other cytokines. IFNγ and IL-4 alone did not affect the adhesion of monocytes. These results show that CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes can be recruited to the inflamed endothelium, but CD14⁺CD16⁺ monocytes adhere to endothelial cells without inflammations twice as strongly as CD14⁺⁺CD16⁻ monocytes.     

The role of Ca<Superscript>2+</Superscript>, H<Superscript>+</Superscript>, and Cl<Superscript>−</Superscript> ions in generation of variation potential in pumpkin plants

The contributions of Ca²⁺, H⁺, and Cl⁻ in generation of variation potentials (VP) in 3- to 4-week-old pumpkin (Cucurbita pepo L., cv. Mozoleevskaya) plants were assessed. During VP generation, transient alkalinization of the medium around the stem was recorded with a potentiometric method. The pH changes were kinetically similar to the electric potential changes and were apparently due to temporal suppression of the plasma-membrane electrogenic H⁺ pump. These data and the observed inhibition of VP in the stem zone treated locally with a metabolic inhibitor (NaN₃) indicate that the VP generation is related to the reversible suppression of the H⁺-pump. The anion channel blocker (ethacrynic acid) decelerated significantly the front slope of VP and reduced the VP amplitude. A short-term increase in external Cl⁻ concentration around the stem was observed during potential transients representing the VP front slope and the pulses integrated into VP. The removal of Ca²⁺ from extracellular medium inhibited the VP generation. It is proposed that Ca²⁺ plays a role in activation of anion channels and in the H⁺-pump inactivation. The VP generation is probably determined by a complex mechanism, with contributions from passive ion fluxes (Ca²⁺, Cl⁻) moving along the electrochemical gradients and from changes in the electrogenic pump activity.     

Succinate is the controller of O<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I : negative modulation by malate,positive by cyanide

Mitochondrial production of H₂O₂ is low with NAD substrates (glutamate/pyruvate, 3 and 2 mM) (G/P) and increases over ten times upon further addition of succinate, with the formation of a sigmoidal curve (semimaximal value at 290 μM, maximal H₂O₂ production at 600 μM succinate). Malate counteracts rapidly the succinate induced increased H₂O₂ release and moves the succinate dependent H₂O₂ production curve to the right. Nitric oxide (NO) and carbon monoxide (CO) are cytochrome c oxidase inhibitors which increase mitochondrial ROS production. Cyanide (CN⁻) was used to mimic NO and CO. In the presence of G/P and succinate (300 μM), CN⁻ progressively increased the H₂O₂ release rate, starting at 1.5 μM. The succinate dependent H₂O₂ production curve was moved to the left by 30 μM CN⁻. The V_max was little modified. We conclude that succinate is the controller of mitochondrial H₂O₂ production, modulated by malate and CN⁻. We propose that succinate promotes an interaction between Complex II and Complex I, which activates O₂⁻ production.     

The Role of H<Superscript>+</Superscript>/OH<Superscript>−</Superscript> Channels in the Salt Stress Response of <Emphasis Type="Italic">Chara australis</Emphasis>

We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl₂. The effects are due to an increase in Na⁺ concentration rather than an increase in Cl⁻ concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575–1591, 2008) described the rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate the shift of membrane potential difference (PD) to levels above −100 mV and the change of shape of the current–voltage (I/V) profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct H⁺ or OH⁻. OH⁻ was chosen, as H⁺ required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline APW stimulated opening of more OH⁻ channels. Recovery was still possible even at a PD near −50 mV, with partial return of proton pumping and a decrease in OH⁻ current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics of OH⁻ channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences of opening of H⁺ or OH⁻ channels while the cell is under salt stress are discussed.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

Negative molecular ion of deuterium D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>

D₂⁻ ions produced in collisions of D⁻ ions with relative energies of 2.5–9.2 eV were detected for the first time. It is shown that the effective cross section for this reaction is no less than 1.5 × 10⁻¹⁴ cm². Along with the theoretically predicted short-lived state of negative molecular deuterium ions, a state existing for more than 1 μs was observed.     

Effect of NO<Subscript>3</Subscript><Superscript>−</Superscript>:NH<Subscript>4</Subscript><Superscript>+</Superscript> ratios on growth,root morphology and leaf metabolism of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) seedlings

The effect of NO₃ ^?:NH₄ ⁺ ratio (14:1, 9:6, 7.5:7.5, 1:14, total 15 mmol/L N) in the nutrient solution on biomass, root morphology, and C and N metabolism parameter in hydroponically grown oilseed rape (Brassica napus L.) was evaluated. The dry weights of leaves and roots were significantly largest at the equal NO₃ ^?:NH₄ ⁺ ratio (7.5:7.5) compared with those of high NO₃ ^?:NH₄ ⁺ ratio (14:1) or low NO₃ ^?:NH₄ ⁺ ratio (1:14). Additionally, low NO₃ ^?:NH₄ ⁺ ratio (1:14) reduced total root length and root surface area compared with the equal NO₃ ^?:NH₄ ⁺ ratio (7.5:7.5), while high NO₃ ^?:NH₄ ⁺ ratio (14:1) did not show any significant effect on root morphology except average diameter. The maximum of chlorophyll a, chlorophyll b and carotenoid were obtained under 7.5:7.5 treatment, whereas the maximum of the leaf net photosynthetic (P _n), stomatal conductance (G _s) and transpiration rate (T _r) were increased with increase in NH₄ ⁺ concentration in the nutrient solution. The activity of nitrate reductase (NR) showed a significant difference at different NO₃ ^?:NH₄ ⁺ ratios and ranged 9:6 > 7.5:7.5 > 14:1 > 1:14, whereas the range of soluble sugar and soluble protein was 7.5:7.5 > 1:14 > 9:6 > 14:1. Our study reveals that oilseed rape growth is greater under 7.5:7.5 treatment than that under three other treatments. Oilseed rape growth at high or low NO₃ ^?:NH₄ ⁺ ratios was inhibited by decreased pigments, NR activity, soluble sugar, and soluble protein, whereas subdued root growth should be apprehended considerate under high NH₄ ⁺ condition.     

Transport of nitrogen and zinc to rhodes grass by arbuscular mycorrhiza and roots as affected by different nitrogen sources (NH<Subscript>4</Subscript><Superscript>+</Superscript>-N and NO<Subscript>3</Subscript><Superscript>−</Superscript>-N)

We investigated the effect of mineral nitrogen forms on transfer of nitrogen (N) and zinc (Zn) from attached compartments to rhodes grass (Chloris gayana) colonised with arbuscular mycorrhizal fungi (AMF). After being pre-cultivated in substrates with adequate nutrient supply and either AMF inoculated (+AM) or left non-inoculated (?AM), rhodes grass was positioned adjacent to an outer compartment holding a similar substrate but applied with labelled nitrogen (¹⁵N) either as ammonium (NH₄ ⁺) or nitrate (NO₃ ^?), and a high supply of Zn (150 mg kg^?1 DS). Plant roots together with fungal mycelium were either allowed to explore the outer compartment (with root access) or only mycorrhizal hyphae were allowed (without root access). Within each access treatment, biomasses of rhodes grass were not significantly affected by AMF inoculation or N form. AMF contribution to plant ¹⁵N uptake was about double in NH₄ ⁺ compared with NO₃ ^?-supplied treatments while the mycorrhizal influence on plant Zn uptake was insignificant. Without root access, the shoot ¹⁵N/Zn concentration ratio was up to ten-fold higher in +AM than –AM treatments and this ratio increase was clearly more pronounced in NH₄ ⁺ than NO₃ ^?-supplied treatments. In conclusion, rhodes grass in symbiosis with the tested AMF acquired more N when supplied with ammonium. Moreover, there is clear indication that although the AMF have transported both nutrients (N and Zn), N was preferentially transferred as compared to Zn. We confirmed that, while rhodes grass is not able to prevent excessive Zn uptake via roots under conditions of high Zn, mycorrhiza is able to avoid excessive Zn supply to the host plant when the fungus alone has access to contaminated patches.     

Kinetics of Energetic O<Superscript>−</Superscript> Ions in the Discharge Plasmas of Water Vapor and H<Subscript>2</Subscript>O-Containing Mixtures

The process of relaxation of energetic O^– ions formed via dissociative attachment of electrons to molecules in the discharge plasmas of water vapor and H₂O: O₂ mixtures in a strong electric field is studied by the Monte Carlo method. The probability of energetic ions being involved in threshold ion–molecular processes is calculated. It is shown that several percent of energetic O^– ions formed via electron attachment to H₂O molecules in the course of plasma thermalization transform into OH^– ions via charge exchange or are destroyed with the formation of free electrons. The probabilities of charge exchange of O^– ions and electron detachment from them increase significantly (up to 90%) when O^– ions are formed via electron attachment to O₂ molecules in water vapor with an oxygen additive. This effect decreases with increasing oxygen fraction in the mixture but remains appreciable even when the fraction of H₂O molecules in the H₂O: O₂ mixture does not exceed several percent.     

Anionic Selectivity Sequence of the Cl<Superscript>−</Superscript>–H<Superscript>+</Superscript> Symporter in the Synaptosomal Preparation from Rat Brain Cortex

The Na⁺/H⁺ exchanger has been the only unequivocally demonstrated H⁺-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl⁻–H⁺ symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl⁻–H⁺ symporter is NO₃⁻ > Br⁻ > Cl⁻ >> I⁻ = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 μM inhibits the gluconate-dependent alkalinization by 30 ± 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization. Special issue article in honor of Dr. Ricardo Tapia.     

Comparative properties of sensitive to GABA<Subscript>A</Subscript>-ergic ligands Cl<Superscript>−</Superscript>, HCO<Subscript>3</Subscript><Superscript>−</Superscript>-activated Mg<Superscript>2+</Superscript>-ATPase from brain plasma membranes of fish and rats

Action of Cl^? + HCO₃ ^?1 ions on Mg²⁺-ATPase from brain plasma membranes of fish and rats has been studied. Maximal effect of the anions on the “basal” Mg²⁺-ATPase activity is revealed in the presence of 10 mM Cl^? and 3 mM HCO₃ ^?1 at physiological values of pH of incubation medium. The studied Cl^?, HCO₃ ^?-activated Mg²⁺-ATPases of both animal species, by their sensitivity to SH-reagents (5,5-dithio-bis-nitrobenzoic acid, N-ethylmaleimide), oligomycin, and orthovanadate, are similar to transport ATPase of the P-type, but differ from them by molecular properties and by sensitivity to ligands of GABA_A-receptors. It has been established that the sensitive to GABA_A-ergic ligands, Cl^?, HCO₃ ^?-activated Mg²⁺-ATPase from brain of the both animal species is protein of molecular mass around 300 kDa and of Stock’s radius 5.4 nm. In fish the enzyme is composed of one major unit of molecular mass approximately 56 kDa, while in rats-of three subunits of molecular masses about 57, 53, and 45 kDa. A functional and structural coupling of the ATP-hydrolyzing areas of the studied enzyme to sites of binding of GABA_A-receptor ligands is suggested.     

The ambiguous role of the Na<Superscript>+</Superscript>–H<Superscript>+</Superscript> exchanger isoform 1 (NHE1) in leptin-induced oxidative stress in human monocytes

Leptin, a 16-kDa cytokine produced mainly by the adipose tissue, is known to increase energy expenditure while at the same time lowering food intake by acting directly on the hypothalamus. ObRb, the leptin receptor mostly involved in intracellular signaling, is expressed in a wide range of tissues, thus allowing leptin to affect a much broader diversity of biological processes. High concentrations of leptin are encountered in patients with hyperleptinemia, a condition which very often accompanies obesity and which is a direct result of leptin resistance. In the present study, moderate and high concentrations of leptin (16 and 160 ng/ml) were mostly utilized in order to investigate the role of this cytokine in oxidative stress levels in human monocytes. Leptin was found to increase oxidative species production as measured with 2′,7′-dichlorodihydrofluorescein diacetate (general marker of oxidative species, but not O₂^−.) and dihydroethidium (marker of O₂^−.). Surprisingly, it also augmented superoxide dismutase activity. Inhibition of the Na⁺–H⁺ exchanger isoform 1 (NHE1) also inhibited leptin-induced superoxide anion production but at the same time amplified leptin-induced production of other oxidative species. Signaling proteins such as phosphoinositide 3 kinase and conventional isoforms of protein kinase C (α-, β_i-, β_ii-), as well as NADPH oxidase, also participated in leptin signaling. Finally, leptin was found to increase glutathionylation levels of NHE1-bound heat shock protein 70 kDa (Hsp70) but not Hsp70 binding to NHE1.