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.     

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.     

A Voltage-Dependent Ca<Superscript>2+</Superscript> Influx Pathway Regulates the Ca<Superscript>2+</Superscript>-Dependent Cl<Superscript>−</Superscript> Conductance of Renal IMCD-3 Cells

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca²⁺-dependent Cl⁻ current (I_CLCA). Here we report that I_CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al³⁺-sensitive, voltage-dependent, Ca²⁺ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), I_CLCA was found to be inactive and resting currents were predominantly K⁺ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I_CLCA (T _0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in I_CLCA (T _0.5 ~ 100 s). The activation of I_CLCA by depolarization was reduced by lowering extracellular Ca²⁺ and completely inhibited by buffering cytosolic Ca²⁺ with EGTA, suggesting a role for Ca²⁺ influx in the activation of I_CLCA. However, raising bulk cytosolic Ca²⁺ at −60 mV did not produce sustained I_CLCA activity. Therefore I_CLCA is dependent on both an increase in intracellular Ca²⁺ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca²⁺ influx pathway that displays equal permeability to Ca²⁺ and Ba²⁺ ions and that is blocked by extracellular Al³⁺ and La³⁺. Furthermore, Al³⁺ completely and reversibly inhibited depolarization-induced activation of I_CLCA, thereby directly linking Ca²⁺ influx to activation of I_CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I_CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Molecular Weight and Subunit Composition of the Sensitive to GABA-Ergic Ligands Cl<Superscript>−</Superscript>/HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-Stimulated Mg<Superscript>2+</Superscript>-ATPase from Plasma Membrane of Rat Brain

The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases.     

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).     

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.     

Synthesis of the β-amyloid fragment <Superscript>5</Superscript>RHDSGY<Superscript>10</Superscript> and its isomers

The peptide RHDSGY, a fragment of the human β-amyloid Zn-binding site, and its isomers RH(D-Asp)SGY and RH(β-Asp)SGY have been obtained as amides by means of solid-phase synthesis and analyzed by HPLC and various mass spectrometric methods. The problem of low yield of the RHDSGY peptide and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of such side-products as RH(β-Asp)SGY (or RHDSGY during synthesis of RH(β-Asp)SGY) and RH(Asp-imide) SGY was solved via selection of individual reagents for removal of Fmoc groups from α-amino groups of the growing peptide chain.     

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.     

H<Superscript>N</Superscript>, N,C<Superscript>α</Superscript> and C<Superscript>β</Superscript> assignments of the two periplasmic domains of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> DsbD

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H^N, N, C^α and C^β) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using ¹³C^αβ shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.     

Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> antiport as a possible mechanism of the Ca<Superscript>2+</Superscript>-translocating ATPase functioning in vesicles of bean root nodule’s symbiosome membrane

Using vesicles of symbiosome membrane (SM), it was shown that the Ca²⁺-ATPase can function as an ATP-energized Ca²⁺/H⁺ antiporter. The initial rate of the acidic shift inside the vesicles, as well as the rate of the ITP-dependent alkalization of the medium inside them markedly increased in the presence of valinomycin. This process was rapidly stopped by eosin Y, a known inhibitor of the type IIB Ca²⁺-ATPase. ITP-dependent uptake of Ca²⁺ was blocked after the addition to the reaction mixture of nigericin in the presence of K⁺. Under these conditions, the alkaline shift of pH inside the vesicles occurred, leading to the inhibition of operation of the calcium pump in SM. Evaluation of the pH shifts inside the vesicles by using pH-indicator pyranine confirmed the ion-exchange mechanism of the Ca²⁺-ATPase functioning in the SM.     

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      

Effect of different methods of Ca<Superscript>2+</Superscript> extraction from PSII oxygen-evolving complex on the Q<Subscript>A</Subscript><Superscript>−</Superscript> oxidation kinetics

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn₄CaO₅ of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca²⁺ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca²⁺ extraction from OEC on the kinetics of the reduced primary electron acceptor (Q_A ^?) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from Q_A ^? to the secondary quinone acceptor Q_B. Electron transfer from Q_A ^? to Q_B in photosystem II membranes with an occupied Q_B site was slowed down by a factor of 8. However, addition of EGTA or CaCl₂ to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of Q_A ^? oxidation by Q_B in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca²⁺ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the Q_A ^? to Q_B electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca²⁺ depletion.     

<Superscript>2</Superscript>H–<Superscript>13</Superscript>C correlation solid-state NMR for investigating dynamics and water accessibilities of proteins and carbohydrates

Site-specific determination of molecular motion and water accessibility by indirect detection of ²H NMR spectra has advantages over dipolar-coupling based techniques due to the large quadrupolar couplings and the ensuing high angular resolution. Recently, a Rotor Echo Short Pulse IRrAdiaTION mediated cross polarization (^RESPIRATIONCP) technique was developed, which allowed efficient transfer of ²H magnetization to ¹³C at moderate ²H radiofrequency field strengths available on most commercial MAS probes. In this work, we investigate the ²H–¹³C magnetization transfer characteristics of one-bond perdeuterated CD _n spin systems and two-bond H/D exchanged C–(O)–D and C–(N)–D spin systems in carbohydrates and proteins. Our results show that multi-bond, broadband ²H–¹³C polarization transfer can be achieved using ²H radiofrequency fields of ~50 kHz, relatively short contact times of 1.3–1.7 ms, and with sufficiently high sensitivity to enable 2D ²H–¹³C correlation experiments with undistorted ²H spectra in the indirect dimension. To demonstrate the utility of this ²H–¹³C technique for studying molecular motion, we show ²H–¹³C correlation spectra of perdeuterated bacterial cellulose, whose surface glucan chains exhibit a motionally averaged C6 ²H quadrupolar coupling that indicates fast trans-gauche isomerization about the C5–C6 bond. In comparison, the interior chains in the microfibril core are fully immobilized. Application of the ²H–¹³C correlation experiment to H/D exchanged Arabidopsis primary cell walls show that the O–D quadrupolar spectra of the highest polysaccharide peaks can be fit to a two-component model, in which 74% of the spectral intensity, assigned to cellulose, has a near-rigid-limit coupling, while 26% of the intensity, assigned to matrix polysaccharides, has a weakened coupling of 50 kHz. The latter O–D quadrupolar order parameter of 0.22 is significantly smaller than previously reported C–D dipolar order parameters of 0.46–0.55 for pectins, suggesting that additional motions exist at the C–O bonds in the wall polysaccharides. ²H–¹³C polarization transfer profiles are also compared between statistically deuterated and H/D exchanged GB1.     

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.     

β<Subscript>2</Subscript>-Integrin-Mediated Adhesion and Intracellular Ca<Superscript>2+</Superscript> Release in Human Eosinophils

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO₃-buffered HybriCare medium maintained in a humidified 5% CO₂ incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca²⁺] that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca²⁺] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β₂-integrins, or jasplakinolide (2 μM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 μM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca²⁺] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.