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.     

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.     

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      

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.     

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.     

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.     

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.     

Variability and directionality of temporal changes in δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N of aquatic invertebrate primary consumers

Seasonal oscillations in the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope signatures of aquatic algae can cause seasonal enrichment–depletion cycles in the isotopic composition of planktonic invertebrates (e.g., copepods). Yet, there is growing evidence that seasonal enrichment–depletion cycles also occur in the isotope signatures of larger invertebrate consumers, taxa used to define reference points in isotope-based trophic models (e.g., trophic baselines). To evaluate the general assumption of temporal stability in non-zooplankton aquatic invertebrates, δ¹³C and δ¹⁵N time series data from the literature were analyzed for seasonality and the influence of biotic (feeding group) and abiotic (trophic state, climate regime) factors on isotope temporal patterns. The amplitude of δ¹³C and δ¹⁵N enrichment–depletion cycles was negatively related to body size, although all size-classes of invertebrates displayed a winter-to-summer enrichment in δ¹³C and depletion in δ¹⁵N. Among feeding groups, periphytic grazers were more variable and displayed larger temporal changes in δ¹³C than detritivores. For nitrogen, temporal variability and magnitude of directional change of δ¹⁵N was most strongly related to ecosystem trophic state (eutrophic > mesotrophic, oligotrophic). This study provides evidence of seasonality in the isotopic composition of aquatic invertebrates across very broad geographical and ecological gradients as well as identifying factors that are likely to modulate the strength and variability of seasonality. These results emphasize the need for researchers to recognize the likelihood of temporal changes in non-zooplankton aquatic invertebrate consumers at time scales relevant to seasonal studies and, if present, to account for temporal dynamics in isotope trophic models.     

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.     

A <Superscript>13</Superscript>C-detected <Superscript>15</Superscript>N double-quantum NMR experiment to probe arginine side-chain guanidinium <Superscript>15</Superscript>N<Superscript>η</Superscript> chemical shifts

Arginine side-chains are often key for enzyme catalysis, protein–ligand and protein–protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine ¹³C^ζ-detected NMR experiment in which a double-quantum coherence involving the two ¹⁵N^η nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two ¹⁵N^η; this new approach is shown to eliminate the previously deleterious line broadenings of ¹⁵N^η resonances caused by the partially restricted rotation about the C^ζ–N^ε bond. Consequently, sharp and well-resolved ¹⁵N^η resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine ¹⁵N^η nuclei becomes possible using the double-quantum experiment.     

Improved accuracy in measuring one-bond and two-bond <Superscript>15</Superscript>N,<Superscript>13</Superscript>C<Superscript>α</Superscript> coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

An extension to HN(CO-α/β-N,C^α-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants ¹ J _{N′(i)Cα(i)}, ² J _HN(i)Cα(i), ² J _{Cα(i−1)N′(i)}, and ³ J _{Cα(i−1)HN(i)} in ¹⁵N,¹³C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the ² J _CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both ² J _CαN′ and ¹ J _N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

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.     

Measurement of imino <Superscript>1</Superscript>H–<Superscript>1</Superscript>H residual dipolar couplings in RNA

Imino ¹H–¹⁵N residual dipolar couplings (RDCs) provide additional structural information that complements standard ¹H–¹H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino ¹H–¹H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA^Val using a BEST-Jcomp-HMQC2 experiment. ¹H–¹H RDCs are observed between the imino protons in G–U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino ¹H–¹H RDCs complement standard ¹H–¹⁵N RDCs because the ¹H–¹H vectors generally point along the helical axis, roughly perpendicular to ¹H–¹⁵N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the standard experiment. The ability to measure imino ¹H–¹H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NO<Subscript>3</Subscript><Superscript>−</Superscript>-Driven SO<Subscript>4</Subscript><Superscript>2−</Superscript> Production in Freshwater Ecosystems: Implications for N and S Cycling

Massive anthropogenic acceleration of the global nitrogen (N) cycle has stimulated interest in understanding the fate of excess N loading to aquatic ecosystems. Nitrate (NO₃ ⁻) is traditionally thought to be removed mainly by microbial respiratory denitrification coupled to carbon (C) oxidation, or through biomass assimilation. Alternatively, chemolithoautotrophic bacterial metabolism may remove NO₃ ⁻ by coupling its reduction with the oxidation of sulfide to sulfate (SO₄ ²⁻). The NO₃ ⁻ may be reduced to N₂ or to NH₄ ⁺, a form of dissimilatory nitrate reduction to ammonium (DNRA). The objectives of this study were to investigate the importance of S oxidation as a NO₃ ⁻ removal process across diverse freshwater streams, lakes, and wetlands in southwestern Michigan (USA). Simultaneous NO₃ ⁻ removal and SO₄ ²⁻ production were observed in situ using modified “push-pull” methods in nine streams, nine wetlands, and three lakes. The measured SO₄ ²⁻ production can account for a significant fraction (25–40%) of the overall NO₃ ⁻ removal. Addition of ¹⁵NO₃ ⁻ and measurement of ¹⁵NH₄ ⁺ production using the push–pull method revealed that DNRA was a potentially important process of NO₃ ⁻ removal, particularly in wetland sediments. Enrichment cultures suggest that Thiomicrospira denitrificans may be one of the organisms responsible for this metabolism. These results indicate that NO₃ ⁻-driven SO₄ ²⁻ production could be widespread and biogeochemically important in freshwater sediments. Removal of NO₃ ⁻ by DNRA may not ameliorate problems such as eutrophication because the N remains bio-available. Additionally, if sulfur (S) pollution enhances NO₃ ⁻ removal in freshwaters, then controls on N processing in landscapes subject to S and N pollution are more complex than previously appreciated. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Accurate measurement of <Superscript>15</Superscript>N–<Superscript>13</Superscript>C residual dipolar couplings in nucleic acids

New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ ¹H–¹³C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H₁–C₁–N_1/9 (MQ-HCN-QJ) or H_6/8–C_6/8–N_1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and 1/(2J_NC) (attenuated spectrum). The different types of ¹⁵N–¹³C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time ¹⁵N evolution period and the offset of two frequency-selective ¹³C pulses. The methods are demonstrated for a uniformly ¹³C, ¹⁵N-enriched 24-nucleotide stem-loop RNA sequence, helix-35, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35 sample result in an estimated precision of ca. 0.07 Hz for ¹D_NC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35, refined using independently measured observables, including ¹³C–¹H, ¹³C–¹³C and ¹H–¹H dipolar couplings.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0646-2.     

Carbonyl carbon label selective (CCLS) <Superscript>1</Superscript>H–<Superscript>15</Superscript>N HSQC experiment for improved detection of backbone <Superscript>13</Superscript>C–<Superscript>15</Superscript>N cross peaks in larger proteins

We present a highly sensitive pulse sequence, carbonyl carbon label selective ¹H–¹⁵N HSQC (CCLS-HSQC) for the detection of signals from ¹H–¹⁵N units involved in ¹³C′–¹⁵N linkages. The CCLS-HSQC pulse sequence utilizes a modified ¹⁵N CT evolution period equal to 1/( ) (∼33 ms) to select for ¹³C′–¹⁵N pairs. By collecting CCLS-HSQC and HNCO data for two proteins (8 kDa ubiquitin and 20 kDa HscB) at various temperatures (5–40°C) in order to vary correlation times, we demonstrate the superiority of the CCLS-HSQC pulse sequence for proteins with long correlation times (i.e. higher molecular weight). We then show that the CCLS-HSQC experiment yields assignments in the case of a 41 kDa protein incorporating pairs of ¹⁵N- and ¹³C′-labeled amino acids, where a TROSY 2D-HN(CO) had failed. Although the approach requires that the ¹H–¹⁵N HSQC cross peaks be observable, it does not require deuteration of the protein. The method is suitable for larger proteins and is less affected by conformational exchange than HNCO experiments, which require a longer period of transverse ¹⁵N magnetization. The method also is tolerant to the partial loss of signal from isotopic dilution (scrambling). This approach will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large enzymes, and partially folded or unfolded proteins.