A dual 13C and 15N long-term labelling technique to investigate uptake and translocation of C and N in beech (Fagus sylvatica L.)

A continuous dual 13CO2 and 15NH4(15)NO3 labelling experimental set-up is presented that was used to investigate the C and N uptake and allocation within 3-year old beech (Fagus sylvatica L.) during one growing season. The C and N allocation pattern was determined after six, twelve and eighteen weeks of growth. The carbon uptake was distinctly different in the three phases examined: The first six weeks after budbreak were dedicated to leaf growth with a R/S (root to shoot) ratio of 0.14 for the new carbon. The second growth phase showed a balanced R/S ratio of C allocation and after week 13, the root compartment was the main carbon sink (R/S = 6.97). Nitrogen allocation was more basipetal as compared to carbon. In the second growth phase, R/S of Nnew was 5.57 but fell to 3.54 for the third growth phase probably due to formation of reserves in buds and stem.     

Triple resonance experiments for aligned sample solid-state NMR of (13)C and (15)N labeled proteins

Initial steps in the development of a suite of triple-resonance (1)H/(13)C/(15)N solid-state NMR experiments applicable to aligned samples of (13)C and (15)N labeled proteins are described. The experiments take advantage of the opportunities for (13)C detection without the need for homonuclear (13)C/(13)C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are approximately 20% randomly labeled with (13)C in all backbone and side chain carbon sites and approximately 100% uniformly (15)N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are (13)C labeled at only the alpha-carbon and (15)N labeled at the amide nitrogen of a few residues. The requirement for homonuclear (13)C/(13)C decoupling while detecting (13)C signals is avoided in the first case because of the low probability of any two (13)C nuclei being bonded to each other; in the second case, the labeled (13)C(alpha) sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the (13)C chemical shift and (1)H-(13)C and (15)N-(13)C heteronuclear dipolar coupling frequencies associated with the (13)C(alpha) and (13)C' backbone sites, which provide orientation constraints complementary to those derived from the (15)N labeled amide backbone sites. (13)C/(13)C spin-exchange experiments identify proximate carbon sites. The ability to measure (13)C-(15)N dipolar coupling frequencies and correlate (13)C and (15)N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the (13)C chemical shift and (1)H-(13)C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.     

Stable carbon and nitrogen isotope signatures of root-holoparasitic Cynomorium songaricum and its hosts at the Tibetan plateau and the surrounding Gobi desert in China

We first measured the δ(13)C and δ(15)N values of root holoparasite Cynomorium songaricum and its hosts from 19 sites across four provinces in northwest China, in an attempt to investigate their nutritional relationship at the Tibetan plateau and the surrounding Gobi desert. Our study showed that the δ(13)C of C. songaricum closely mirrored the values of its hosts, Nitraria tangutorum and N. sibirica across all sampling sites. C. songaricum was significantly depleted in (13)C compared to host plants at the Tibetan plateau, showing an average parasite/host δ(13)C difference of-0.6?‰. In contrast, (15)N of C. songaricum was significantly enriched by+1.3?‰ compared to the hosts, implying that these holoparasites had other nitrogen resources. Although no difference in the δ(13)C and δ(15)N values between holoparasites and hosts was detected, the δ(13)C and δ(15)N values of holoparasites were significantly correlated with those of their hosts at the Gobi desert. The δ(13)C versus δ(15)N values were significantly but negatively correlated for the hosts; however, holoparasite/host variation in δ(13)C was not correlated with the variation in δ(15)N. The δ(13)C versus δ(15)N values were negatively correlated in C. songaricum, and this relationship tended to be magnified along the increasing elevations independent of the host plants. C. songaricum at the Tibetan plateau exhibited different δ(13)C and δ(15)N signatures compared with those at the Gobi desert. Furthermore, both δ(13)C and δ(15)N values of C. songaricum and its host plants in salt marshes at the Tibetan plateau were different from those in sand sites at the Tibetan plateau and the Gobi desert. Our results indicate that the isotopic difference depends on the different altitudes and habitats and is host-specific.     

Double-quantum filtered rotational-echo double resonance

The homonuclear scalar coupling of a directly bonded 13C-13C pair has been used to create a double-quantum filter (DQF) to remove the natural-abundance 13C background in 13C{15N} rotational-echo double-resonance (REDOR) experiments. The DQF scalar and REDOR dipolar evolution periods are coincident which is important for sensitivity in the event of weak 13C-15N dipolar coupling. Calculated and observed 13C{15N} DQF-REDOR dephasings were in agreement for a test sample of mixed recrystallized labeled alanines. Glycine metabolism in a single uniform-15N soybean leaf labeled for 6 min by 13CO2 was measured quantitatively by 13C{15N} DQF-REDOR with no background interferences.     

The first in vivo observation of (13)C-(15)N coupling in mammalian brain.

[5-(13)C,(15)N]Glutamine, with (1)J((13)C-(15)N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by (13)C MRS at 4.7 T. The brain [5-(13)C]glutamine peak consisted of the doublet from [5-(13)C,(15)N]glutamine and the center [5-(13)C,(14)N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-(13)C,(15)N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-(13)C,(15)N]glutamine was formed by glial uptake of released neurotransmitter [5-(13)C]glutamate and its reaction with (15)NH(3) catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively (13)C-enriched by intravenous [2,5-(13)C]glucose infusion to (13)C-label whole-brain glutamate C5, followed by [(12)C]glucose infusion to chase (13)C from the small and rapidly turning-over glial glutamate pool, leaving (13)C mainly in the neurotransmitter [5-(13)C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-(13)C,(15)N]glutamine arises from a coupling between (13)C of neuronal origin and (15)N of glial origin. Measurement of the rate of brain [5-(13)C,(15)N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

Helicobacter pylori in vivo diagnosis after food intake: results of simultaneous [15N]urea urine and [13C]acetate breath tests

The protocols for 13C and 15N H. pylori tests stipulate that the diagnostic agent should be taken on an empty stomach. It is presumed that food intake prior to the tests leads to less reliable test results due to a prolongation of the gastric residence time of the diagnostic agent urea. This might allow the bacteria to split a higher proportion of urea, resulting in an increased number of false positives. 12 probands received 150mg [15N]urea and 75 mg sodium [13C]acetate in 75 ml orange juice as a test drink. [15N]Urea served as an agent to diagnose gastric H. pylori colonization. The 15N tests were evaluated using a urine sample of the second hour after test start. [13C]Acetate served as a marker of the gastric emptying of water-soluble food including the urea under the influence of food intake. Breath air samples were taken to calculate the gastric emptying half life (EHL) and the apparent resorption time (RT) of the urea. The double tests were carried out four times within four weeks using identical test protocols but different standardized time periods of pretest fasting: overnight, two hours prior to test, one hour prior to test, and no fasting at all. The food intake amount was standardized. Five probands testing positive in the overnight fasting test were also found to be positive in the other test variants with more or less empty stomachs. Seven other probands testing negative after overnight fasting tested negative in the other test variants as well. It is concluded that food intake prior to the test drink does not have much of an influence on the gastric residence time of urea and so on the qualitative H. pylori test results. Due to identical behaviour of [13C]urea and [15N]urea in the stomach, this influence is believed to be independent on the labelling isotope. For survey purpose, no fasting conditions are required for the H. pylori tests.     

Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the alpha-spectrin SH3 domain

Future structural investigations of proteins by solid-state CPMAS NMR will rely on uniformly labeled protein samples showing spectra with an excellent resolution. NMR samples of the solid alpha-spectrin SH3 domain were generated in four different ways, and their (13)C CPMAS spectra were compared. The spectrum of a [u-(13)C, (15)N]-labeled sample generated by precipitation shows very narrow (13)C signals and resolved scalar carbon-carbon couplings. Linewidths of 16-19 Hz were found for the three alanine C(beta )signals of a selectively labeled [70% 3-(13)C]alanine-enriched SH3 sample. The signal pattern of the isoleucine, of all prolines, valines, alanines, and serines, and of three of the four threonines were identified in 2D (13)C-(13)C RFDR spectra of the [u-(13)C, (15)N]-labeled SH3 sample. A comparison of the (13)C chemical shifts of the found signal patterns with the (13)C assignment obtained in solution shows an intriguing match.     

Effects of N-application on utilization of 15N and 13C and quality in two wheat cultivars

We studied N uptake and distribution in wheat, and the incorporation of nitrogen and carbon into gluten and non-gluten proteins using a double-labelling approach with 15N and 13C. Doses of N-fertilizer were split and applied at emergence, onset of stem elongation, and heading at rates of 280/140/140 mg N pot(-1), respectively simulating 90/45/45 kg N ha(-1). Five different combinations of N-fertilizations containing no or 10 % 15N were performed. The recovery of 15N added at the stages emergence, stem elongation or heading were 42, 60, and 64 %. Application of 15N at all three stages yielded in 51 % recovery. Remobilisation of straw N was greater for Golia. The 15N concentration in gluten proteins of Golia show higher values than Gonen. The ratio of 15N gluten/15N non-gluten proteins of Golia were higher, which implies a lower non-gluten protein activity during grain filling. The 13C concentration in gluten and non-gluten proteins did not differ between both cultivars.     

Regional variation and relationships between the contaminants dde and selenium and stable isotopes in swallows nesting along the Rio Grande and one reference site, Texas, USA

Cave swallows (Petrochelidon fulva) and cliff swallows (P. pyrrhonota) nest in numerous colonies throughout the Texas portion of the Rio Grande along the U.S. border with Mexico. We collected swallows during 1999 and 2000 from eight locations along the Rio Grande to determine if delta15N and delta13C values could be used to predict 1,1-di-(p-chlorophenyl-)2,2-dichloroethene (DDE) and selenium (Se) contaminant burdens in insectivorous birds nesting across a geographic gradient in the Texas-Mexico border and to discern if stable isotopes could help discriminate between local versus nonlocal acquisition of contaminants.We analysed delta15N and delta13C in liver and muscle and DDE and Se in swallow carcasses. Within individuals, delta15N was higher in liver than in muscle of both species by an average of 1.34%, whereas delta13C was 0.145% higher in muscle than in liver. Significant differences occurred among locations in delta15N and delta13C values in liver and muscle of both species. Cave swallows from three locations in the Lower Rio Grande Valley were more enriched in delta15N than swallows from other sites. In general, swallows nesting in more northern latitudes along the Rio Grande had lower delta15N and delta13C values than those nesting farther south. Concentrations of DDE were significantly greater in swallows from El Paso, Llano Grande, and Pharr than in those from Brownsville, Falcon Lake, Laredo, Del Rio, and a reference site outside the Rio Grande. All swallows (n = 21) from El Paso, Llano Grande, and Pharr had DDE concentrations > or = 3 microg g(-1) wet weight (ww), a value three times greater than the estimated threshold in avian prey that could cause potential reproductive failures in raptors. Concentrations of Se also were significantly greater in El Paso and Del Rio than at other locations. Most Se concentrations were not of concern for direct effects on birds or their predators. Principal component analysis indicated some positive correlations between delta15N and delta13C values in tissues and contaminant concentrations in carcass; however, analysis of covariance suggested a stronger effect of location on concentrations of DDE and Se. At the local level (Llano Grande and Pharr) there was a significant positive correlation between delta15N in liver and DDE concentrations in swallow carcasses; however, Se concentrations were not significantly correlated with isotopes even at the local level. Our results provide a good database of delta15N and delta13C values for insectivorous birds nesting along the Rio Grande. Similar ecologies for cave and cliff swallows and their abundance and wide distribution along the Rio Grande make them ideal indicators of environmental pollution of this portion of the Texas-Mexico border.     

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.     

A solid-state NMR application of the anomeric effect in carbohydrates: galactosamine, glucosamine, and N-acetyl-glucosamine

Simple 2D 13C/15N heteronuclear correlation solid-state NMR spectroscopy was implemented to resolve the 15N resonances of the alpha and beta anomers of three amino monosaccharides: galactosamine (GalN), glucosamine hydrochloride (GlcN), and N-acetyl-glucosamine (GlcNAc) labeled specifically with 13C1/15N spin pairs. Although the 15N resonances could not be distinguished in normal 1D spectra, they were well resolved in 2D double CP/MAS correlation spectra by taking advantage of the 13C spectral resolution. The alpha and beta resonances shifted apart by 3-5 ppm in their 13C chemical shifts, and differed by 1-2 ppm in the extended 15N dimension. Aside from this, the detection of other 13C/15N correlations over short distances was also achieved arising from the C2, C3 and CO carbons present in natural abundance. 2D double CP/MAS chemical shift correlation NMR spectroscopy is a simple and powerful technique to characterize the anomeric effect of amino monosaccharides. Applications of the 2D method reveal well-resolved 15N and 13C chemical shifts might be useful for structural determination on carbohydrates of biological significance, such as glycopeptide or glycolipids.     

Coherence selection in double CP MAS NMR spectroscopy

Applications of double cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy, via (1)H/(15)N and then (15)N/(13)C coherence transfers, for (13)C coherence selection are demonstrated on a (15)N/(13)C-labeled N-acetyl-glucosamine (GlcNAc) compound. The (15)N/(13)C coherence transfer is very sensitive to the settings of the experimental parameters. To resolve explicitly these parameter dependences, we have systematically monitored the (13)C{(15)N/(1)H} signal as a function of the rf field strength and the MAS frequency. The data reveal that the zero-quantum coherence transfer, with which the (13)C effective rf field is larger than that of the (15)N by the spinning frequency, would give better signal sensitivity. We demonstrate in one- and two-dimensional double CP experiments that spectral editing can be achieved by tailoring the experimental parameters, such as the rf field strengths and/or the MAS frequency.     

The effect of soil temperature and moisture on organic matter decomposition and plant growth

The effect of soil temperature and moisture on plant growth and mineralisation of organic residues was investigated using 15N-labelled soybean residues and temperature-controlled tanks in the glasshouse. Treatments were arranged in a factorial design with: three soil temperatures (20, 26 and 30 degrees C), two soil moisture regimes (8% (-800 Kpa) or 12% (-100 Kpa)), soybean residues added (enriched at 1.82 atom % 15N excess) or no residues; and either sown with ryegrass or not sown. Pots were sampled six weeks after planting and 15N-enrichment and delta13C of the plant and soil fractions were determined. Soil inorganic N was also periodically measured. Available inorganic N increased significantly with addition of residues and generally decreased with increasing temperature. Plant dry matter decreased significantly with increase in soil temperature and increased with increasing moisture. Root-to-shoot ratio declined with increased temperature and moisture. Percentage nitrogen derived from residues (%Ndfr) increased linearly with increased temperature and moisture. Delta13C decreased linearly with increasing temperature and decreasing moisture status. There was a significant correlation between transpiration and dry matter production, but there was no correlation between water use efficiency and delta13C. The results suggest that C: N ratio of the root material effects the root turnover and in turn the water supply capacity of the root system.     

Three-dimensional 13C shift/1H-15N coupling/15N shift solid-state NMR correlation spectroscopy.

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly 13C- and 15N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from 1H to 13C and from 13C to 15N with flip-flop (phase and frequency switched) Lee-Goldburg irradiation for both 13C homonuclear decoupling and 1H-15N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields 13C shift/1H-15N coupling/15N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate 13C and 15N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly 13C- and 15N-labeled proteins.     

Triple resonance solid state NMR experiments with reduced dimensionality evolution periods

Two solid state NMR triple resonance experiments which utilize the simultaneous incrementation of two chemical shift evolution periods to obtain a spectrum with reduced dimensionality are described. The CO N CA experiment establishes the correlation of (13)C(i-1) to (13)C alpha(i) and (15)N(i) by simultaneously encoding the (13)CO(i-1) and (15)N(i) chemical shifts. The CA N COCA experiment establishes the correlation (13)Ca(i) and (15)CO(i) to (13)C alpha(i-1) and (15)N(i-1) within a single experiment by simultaneous encoding of the (13)C alpha(i) and (15)N(i) chemical shifts. This experiment establishes sequential amino acid correlations in close analogy to the solution state HNCA experiment. Reduced dimensionality 2D experiments are a practical alternative to recording multiple 3D data sets for the purpose of obtaining sequence-specific resonance assignments of peptides and proteins in the solid state.     

Side-chain H and C resonance assignment in protonated/partially deuterated proteins using an improved 3D(13)C-detected HCC-TOCSY

We propose the use of (13)C-detected 3D HCC-TOCSY experiments for assignment of (1)H and (13)C resonances in protonated and partially deuterated proteins. The experiments extend 2D C-13-start and C-13-observe TOCSY type experiments proposed earlier. Introduction of the third (1)H dimension to 2D TOCSY: (i) reduces the peak overlap and (ii) increases the sensitivity per unit time, even for highly deuterated (>85%) protein samples, which makes this improved method an attractive tool for the side-chain H and C assignment of average sized proteins with natural isotope abundance as well as large partially deuterated proteins. The experiments are demonstrated with a 16 kDa (15)N, (13)C-labeled non-deuterated apo-CcmE and a 48 kDa uniformly (15)N, (13)C-labeled and fractionally ( approximately 90%) deuterated dimeric sFkpA. It is predicted that this method should be suitable for the assignment of methyl (13)C and (1)H chemical shifts of methyl protonated, highly deuterated and (13)C-labeled proteins with even higher molecular weight.     

Correlation of backbone amide and side-chain (13)C resonances in perdeuterated proteins

Side-chain carbon resonance assignments are difficult to obtain for larger proteins. While standard methods require protons for excitation and detection of magnetization, their presence is often unacceptable and often leads to unacceptable relaxation losses at the directly bound carbon sites. In this paper, pulse sequences are presented which provide connectivities between aliphatic side-chain (13)C and amide (1)H and (15)N chemical shifts in fully deuterated, (13)C/(15)N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via (13)C-(13)C isotropic mixing. Direct rather than (13)CO-relayed (15)N-->(13)C(alpha) or (13)C(alpha)-->(15)N transfer steps allow the detection of intraresidual as well as sequential correlations. To avoid ambiguities between these two types in the three-dimensional version of the experiments, a fourth dimension can be introduced to achieve their separation along a (13)C(alpha) frequency axis. The novel methods are demonstrated with the uniformly (2)H/(13)C/(15)N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.     

Short-term changes in delta(13)C and delta(15)N signatures of water discharged from grazed grasslands

The composition of dissolved organic matter (DOM) in a soil is the product of a variety of soil processes. Changes in the composition of DOM in water discharged from soil should, therefore, give an important insight into modifications in these soil processes. We hypothesise that these processes in soils, under different grassland management regimes, would be affected to different extents by the short-term disturbance of a storm event and that evidence of this could be detected in delta(13)C and delta(15)N signatures in drainage and surface runoff waters. During a storm event we collected discharge waters from 1 ha grassland lysimeters, with or without artificial drainage, which received contrasting fertiliser inputs, and delta(13)C and delta(15)N signatures were determined. Changes in (13)C enrichment during the storm event were clearly identifiable, as were differences between plots for (13)C and (15)N, illustrating that this technique has potential to be a useful tool for identifying and investigating short- and long-term changes in soil organic matter dynamics. Copyright 1999 John Wiley & Sons, Ltd.     

The Effect of Diet Quality on δ(13)C and δ(15)N in the Tissues of Locusts, Locusta migratoria L

Abstract Locust nymphs were raised from hatching to adult locusts on either seedling wheat (C(3)) or maize (C(4)), to determine whether relative enrichments/depletions of (15)N and (13)C within body tissues are influenced by diet. The maize contained less hexose sugars and protein per gram than wheat. The isotopic spacing between the food and the whole insect was found to differ between the two diets. The lower quality maize diet showed an overall +5.1‰ enrichment in δ(15)N compared to + 2.8‰ for wheat, possibly due to increased fractionation due to protein recycling. The maize diet resulted in increased depletion in lipid and trehalose and depletion in chitin relative to diet. The results for both δ(15)N and δ(13)C suggest that substrate recycling was occurring on the low quality maize diet. Therefore diet quality determines the enrichment/depletion in δ(15)N and δ(13)C within organisms.     

NMR determination of the torsion angle psi in alpha-helical peptides and proteins: the HCCN dipolar correlation experiment

Several existing methods permit measurement of the torsion angles phi, psi and chi in peptides and proteins with solid-state MAS NMR experiments. Currently, however, there is not an approach that is applicable to measurement of psi in the angular range -20 degree to -70 degree, commonly found in alpha-helical structures. Accordingly, we have developed a HCCN dipolar correlation MAS experiment that is sensitive and accurate in this regime. An initial REDOR driven (13)C'--(15)N dipolar evolution period is followed by the C' to C(alpha) polarization transfer and by Lee--Goldburg cross polarization recoupling of the (13)C(alpha)(1)H dipolar interaction. The difference between the effective (13)C(1)H and (13)C(15)N dipolar interaction strengths is balanced out by incrementing the (13)C--(15)N dipolar evolution period in steps that are a factor of R(R approximately omega(CH)/omega(CN)) larger than the (13)C--(1)H steps. The resulting dephasing curves are sensitive to variations in psi in the angular region associated with alpha-helical secondary structure. To demonstrate the validity of the technique, we apply it to N-formyl-[U-(13)C,(15)N] Met-Leu-Phe-OH (MLF). The value of psi extracted is consistent with the previous NMR measurements and close to that reported in diffraction studies for the methyl ester of MLF, N-formyl-[U-(13)C,(15)N]Met-Leu-Phe-OMe.