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.     

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.     

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.     

Role of unusual CD4<Superscript>+</Superscript> CD28<Superscript>−</Superscript> T cells in acute coronary syndrome

Acute coronary syndrome (ACS) is a group of clinical symptoms that results from complete or partial occlusive thrombus, which is caused by coronary an atherosclerotic plaque rupture or erosion. According to a recent study, CD4⁺ CD28⁻ T cells are found in atherosclerotic plaques and the peripheral circulation blood in patients with ACS, these cells play an important role in plaque ruptures. CD4⁺ CD28⁻ T cells are an unusual subset of helper cells, which expand and have harmful effects in ACS. In this review, we discuss the current issues on the generation of CD4⁺ CD28⁻ T cells and focus on their phenotypic and functional characteristics relevant to the development of cardiovascular events. Targeting the CD4⁺ CD28⁻ T cells subset in ACS could provide novel therapeutic means to prevent acute life-threatening coronary events.     

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.     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

A computer-aided quantum chemical study of the N<Stack><Subscript>15</Subscript><Superscript>−</Superscript></Stack> cluster

Ab initio (RHF, MP2) and Density Functional Theory (DFT) methods have been used to examine six isomers of the N15m cluster with the 6-31+G* basis set. Different from the known odd-numbered anionic N7m, N9m, and N11m clusters, in which the open-chain structures are the most stable species, the most stable N15m isomer is structure 1 (C1), which may be considered as a complex between the fragments cyclic N5m (D5h) and staggered N10 (D2d). The decomposition pathways of structure 2 (CS), containing two aromatic N5 rings connected by a N5 chain, and the open-chain structure 3 (C2v) were studied at the B3LYP/6-31+G* level of theory. Relative energies were refined at the level of B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G*+ZPE (B3LYP/6-31+G*). The barriers for N2 and N5m (D5h) fission reactions for structure 2 are predicted to be 18.2 and 14.2 kcal x mol(-1), respectively. The corresponding N2+N3m fission barrier for structure 3 is predicted to be 11.2 kcal x mol(-1). Supplementary material is available for this article if you access the article at http://dx.doi.org/10.1007/s00894-003-0118-0. A link in the frame on the left on that page takes you directly to the supplementary material. Figure Structure 1 of the N15m cluster, showing bond distances in A and bond angles in degrees     

Transformation of Δ<Superscript>4</Superscript>-3-ketosteroids by free and immobilized cells of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> actinobacterium

9α-Hydroxy derivatives were prepared from 11 steroids of androstane and pregnane series using Rhodococcus erythropolis VKPM Ac-1740 culture with 0.5–10 g/l substrate concentration in the reaction mixture. 9α-Monohydroxylation proceeded regardless of the substituent structure at C17. However, the structure of the steroid molecule influenced the time of complete conversion of the substrate and the yield of the transformation product. 9α-Hydroxy-androstenedione was obtained in 35 h in a yield of 85% when the maximum concentration of androstenedione (AD) was 10 g/l. 9α-Hydroxy-AD was also formed by the actinobacterium cells entrapped in poly(vinyl alcohol) cryogel beads. Nine successive transformation cycles were carried out using immobilized cells at 4.0 g/l concentration of AD in the medium. The yield of 9α-hydroxy-AD formed during six cycles (from two to eight with the duration of each cycle for 22–24 h) was 98%.     

Presence of Ornithine in the Urate-binding α<Subscript>1</Subscript>—<Subscript>2</Subscript>Globulin

THE urate-binding α₁–α₂ globulin has been isolated from human plasma in a highly purified state¹. The protein was purified by DEAE-‘Sephadex’, ammonium sulphate precipitation and semi-preparative Polyacrylamide gel electrophoresis. The urate-binding α₁–α₂ globulin is a rod-shaped glycoprotein, containing 12.1% carbohydrate, with an isoelectric point of 4.6 and a molecular weight of 67,000 ± 4,000. Amino-acid analysis indicated an unknown basic compound which appeared as an extra peak just in front of lysine¹. To identify this compound, high voltage paper electrophoresis has been carried out on a plate electrophoresis apparatus in pyridine-acetate buffer pH 3.5. A spot separated out corresponding to ornithine. Amino-acid analysis on a BC-200 automatic analyser (Bio-Cal Instruments Co., West Germany), with a 54 cm column at 55° C and with 0.35 M sodium citrate buffer, pH 5.28, as elution buffer at a flow-rate of 150 ml./h, showed that ornithine was present. The presence of ornithine in the protein hydrolysate was also verified by gas chromatography/mass spectrometry².     

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 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.     

Impact of two-bond <Superscript>15</Superscript>N–<Superscript>15</Superscript>N scalar couplings on <Superscript>15</Superscript>N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R ₂ ) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings ( ² J _NN ) of the ¹⁵N_ε nuclei and found that the magnitudes of the ² J _NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ² J _NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_ε R ₂ measurements. However, our experimental data show that the expected modulation via two ² J _NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R ₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ² J _NN couplings. Although the so-called recoupling conditions may cause overestimate of R ₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.     

Sequence specific <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a cataract-related variant G57W of human γS-crystallin

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [¹⁵N–¹H]-HSQC spectrum recorded with uniformly ¹³C/¹⁵N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.     

The neural γ<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript> gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ₂α₁β₂α₁β₂ gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ₂α₁β₂α₁β₂ has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ₂ and 2 α₁ subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models—one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2–M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.     

Selective suppression of excipient signals in 2D <Superscript>1</Superscript>H–<Superscript>13</Superscript>C methyl spectra of biopharmaceutical products

While the use of ¹H–¹³C methyl correlated NMR spectroscopy at natural isotopic abundance has been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies, spectral interference from aliphatic excipients remains a significant obstacle to its widespread application. These signals can cause large baseline artifacts, obscure protein resonances, and cause dynamic range suppression of weak peaks in non-uniform sampling applications, thus hampering both traditional peak-based spectral analyses as well as emerging chemometric methods of analysis. Here we detail modifications to the 2D ¹H–¹³C gradient-selected HSQC experiment that make use of selective pulsing techniques for targeted removal of interfering excipient signals in spectra of the NISTmAb prepared in several different formulations. This approach is demonstrated to selectively reduce interfering excipient signals while still yielding 2D spectra with only modest losses in protein signal. Furthermore, it is shown that spectral modeling based on the SMILE algorithm can be used to simulate and subtract any residual excipient signals and their attendant artifacts from the resulting 2D NMR spectra.     

Functional blockade of α<Subscript>5</Subscript>β<Subscript>1</Subscript> integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

Background  

Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α₅β₁ integrin in liver progenitor cells.     

The effect of metal ions on the binding of ethanol to human alcohol dehydrogenase β<Subscript>2</Subscript>β<Subscript>2</Subscript>

Molecular docking simulations were performed in this study to investigate the importance of both structural and catalytic zinc ions in the human alcohol dehydrogenase beta(2)beta(2) on substrate binding. The structural zinc ion is not only important in maintaining the structural integrity of the enzyme, but also plays an important role in determining substrate binding. The replacement of the catalytic zinc ion or both catalytic and structural zinc ions with Cu(2+) results in better substrate binding affinity than with the wild-type enzyme. The width of the bottleneck formed by L116 and V294 in the substrate binding pocket plays an important role for substrate entrance. In addition, unfavorable contacts between the substrate and T48 and F93 prevent the substrate from moving too close to the metal ion. The optimal binding position occurs between 1.9 and 2.4 A from the catalytic metal ion.