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Sample records for 15n labeling study

  1. A study of 15N- 15N and 15N- 13C spin couplings in some 15N labeled mesoionic 1-oxa and 1-thia-2,3,4-triazoles

    NASA Astrophysics Data System (ADS)

    Jaźwiński, J.; Staszewska, O.; Stefaniak, L.; Webb, G. A.

    1996-03-01

    15N- 15N and 15N- 13C spin-spin couplings are reported for seven 15N labeled 1-oxa and 1-thia-2,3,4-triazoles and three sydnonimines. For the former class of compounds the spin-spin coupling data show a close similarity between the N2N3 and N3N4 bonds which had not previously been suspected from chemical shift measurements.

  2. Production of 15N-Labelled Liquid Organic Fertilisers Based on Manure and Crop Residue for Use in Fertigation Studies.

    PubMed

    Martínez-Alcántara, Belén; Martínez-Cuenca, Mary-Rus; Fernández, Carlos; Legaz, Francisco; Quiñones, Ana

    2016-01-01

    Large quantities of crop residue and animal manure from agricultural and livestock activities are annually produced worldwide. With proper management, these residues are potentially valuable sources of plant nutrients, mainly N. Recycling such subproducts in sustainably-based agricultural systems can minimise the use of mineral fertilisers, and hence reduce the potential risk of surface and groundwater pollution. Therefore, the purpose of this study was to obtain (small scale) two liquid labelled-organic fertilisers, an animal- and a vegetal-based organic (AO and VO, respectively) fertiliser, to be used as organic N sources in subsequent fertigation studies. Forage maize (Zea mays L.) grown under 15N-labelled fertiliser supply was used as raw material for VO fertiliser production, and also as 15N-labelled sheep feed to obtain 15N-labelled manure. The labelled faeces fraction was used as raw material for the AO fertiliser. The VO fertiliser was obtained after an acidic and an enzyme-driven hydrolysis. The AO fertiliser was obtained after acidic hydrolysis. The VO liquid fertiliser presented an N concentration of 330 mg·L-1, 85% of total N was organic, while ammonium and nitrate N accounted for 55% and 45% of the mineral nitrogen fraction, respectively. This fertiliser also exhibited high K, Ca and S concentrations and notable values for the remaining macro- and micronutrients. The AO liquid fertiliser had a similar total N concentration (496 mg·L-1, 82% of total N in an organic form) to that of VO, but its mineral N fraction significantly differed, which came in a predominantly (95%) ammonia form. It also had a high content of N, P, K and other macronutrients, and sufficient Fe, Zn, Mn, Cu and B levels, which suggests its suitability as a potential fertiliser. The percentage of 15N enrichment in both VO and AO liquid fertilisers exceeded 2% 15N atom excess, which enabled their use in subsequent assays run to assess nitrogen uptake efficiency.

  3. Nitrogen assimilation and dissimilation by bacteria and benthic microalgae in tidal mudflat sediment in a 15N labeling study

    NASA Astrophysics Data System (ADS)

    Dähnke, K.; Moneta, A.; Veuger, B.; Soetaert, K.; Middelburg, J. J.

    2012-04-01

    In a short-term 15N-labeling experiment, we investigated the changes in relative utilization of reactive nitrogen in tidal flat sediment, focusing on the relative importance of assimilatory versus dissimilatory processes and the role of benthic microalgae therein. 15N-labeled ammonium and nitrate were added separately to homogenized tidal flat sediment, and 15N was subsequently traced into bulk sediment and inorganic nutrients in pore water. Integration of results in an N cycle model allowed us to quantify rates for the major assimilatory and dissimilatory processes in the sediment. Overall, the results indicate that the equilibrium between assimilation and dissimilation in this tidal mudflat is mainly dependent on the nitrogen source: Nitrate is utilized almost exclusively dissimilatory via denitrification, whereas ammonium is rapidly assimilated, with about a quarter of this assimilation due to BMA activity. The major influence of benthic microalgae is on assimilation of ammonium, ceasing BMA activity turns the sediments from a net ammonium sink to a net source. There is little evidence of dissimilative processes like nitrification in undisturbed sediments, but high initial nitrification rates suggest that in a dynamic environment like tidal flats, intense and fast nitrification/denitrification of ammonium is abundant. The driving mechanisms for assimilation or dissimilation accordingly appear to be ruled to a large extent by external physical forcing, with the entire system being capable of rapid shifts following environmental changes. Our combined experimental and model approach reveals that selective removal of labeled compounds takes place for both ammonium and nitrate. Mechanisms remain unclear, but this finding clearly challenges the traditional labeling approach and underscores the need to consider selective uptake in future labeling studies. Ignoring such selective uptake mechanisms will lead to misinterpretation of process rates when these are estimated

  4. Quantitative study on the fate of residual soil nitrate in winter wheat based on a 15N-labeling method

    PubMed Central

    Zhang, Jing-Ting; Wang, Zhi-Min; Liang, Shuang-Bo; Zhang, Ying-Hua; Lu, Lai-Qing; Wang, Run-Zheng

    2017-01-01

    A considerable amount of surplus nitrogen (N), which primarily takes the form of nitrate, accumulates in the soil profile after harvesting crops from an intensive production system in the North China Plain. The residual soil nitrate (RSN) is a key factor that is included in the N recommendation algorithm. Quantifying the utilization and losses of RSN is a fundamental necessity for optimizing crop N management, improving N use efficiency, and reducing the impact derived from farmland N losses on the environment. In this study, a 15N-labeling method was introduced to study the fate of the RSN quantitatively during the winter wheat growing season by 15N tracer technique combined with a soil column study. A soil column with a 2 m height was vertically divided into 10 20-cm layers, and the RSN in each layer was individually labeled with a 15N tracer before the wheat was sown. The results indicated that approximately 17.68% of the crop N derived from RSN was located in the 0–2 m soil profile prior to wheat sowing. The wheat recovery proportions of RSN at various layers ranged from 0.21% to 33.46%. The percentages that still remained in the soil profile after the wheat harvest ranged from 47.08% to 75.44%, and 19.46–32.64% of the RSN was unaccounted for. Upward and downward movements in the RSN were observed, and the maximum upward and downward distances were 40 cm and 100 cm, respectively. In general, the 15N-labeling method contributes to a deeper understanding of the fates of the RSN. Considering the low crop recovery of the RSN from deep soil layers, water and N saving practices should be adopted during crop production. PMID:28170440

  5. Quantitative study on the fate of residual soil nitrate in winter wheat based on a 15N-labeling method.

    PubMed

    Zhang, Jing-Ting; Wang, Zhi-Min; Liang, Shuang-Bo; Zhang, Ying-Hua; Zhou, Shun-Li; Lu, Lai-Qing; Wang, Run-Zheng

    2017-01-01

    A considerable amount of surplus nitrogen (N), which primarily takes the form of nitrate, accumulates in the soil profile after harvesting crops from an intensive production system in the North China Plain. The residual soil nitrate (RSN) is a key factor that is included in the N recommendation algorithm. Quantifying the utilization and losses of RSN is a fundamental necessity for optimizing crop N management, improving N use efficiency, and reducing the impact derived from farmland N losses on the environment. In this study, a 15N-labeling method was introduced to study the fate of the RSN quantitatively during the winter wheat growing season by 15N tracer technique combined with a soil column study. A soil column with a 2 m height was vertically divided into 10 20-cm layers, and the RSN in each layer was individually labeled with a 15N tracer before the wheat was sown. The results indicated that approximately 17.68% of the crop N derived from RSN was located in the 0-2 m soil profile prior to wheat sowing. The wheat recovery proportions of RSN at various layers ranged from 0.21% to 33.46%. The percentages that still remained in the soil profile after the wheat harvest ranged from 47.08% to 75.44%, and 19.46-32.64% of the RSN was unaccounted for. Upward and downward movements in the RSN were observed, and the maximum upward and downward distances were 40 cm and 100 cm, respectively. In general, the 15N-labeling method contributes to a deeper understanding of the fates of the RSN. Considering the low crop recovery of the RSN from deep soil layers, water and N saving practices should be adopted during crop production.

  6. Production of 15N-Labelled Liquid Organic Fertilisers Based on Manure and Crop Residue for Use in Fertigation Studies

    PubMed Central

    Martínez-Alcántara, Belén; Martínez-Cuenca, Mary-Rus; Fernández, Carlos; Legaz, Francisco; Quiñones, Ana

    2016-01-01

    Large quantities of crop residue and animal manure from agricultural and livestock activities are annually produced worldwide. With proper management, these residues are potentially valuable sources of plant nutrients, mainly N. Recycling such subproducts in sustainably-based agricultural systems can minimise the use of mineral fertilisers, and hence reduce the potential risk of surface and groundwater pollution. Therefore, the purpose of this study was to obtain (small scale) two liquid labelled-organic fertilisers, an animal- and a vegetal-based organic (AO and VO, respectively) fertiliser, to be used as organic N sources in subsequent fertigation studies. Forage maize (Zea mays L.) grown under 15N-labelled fertiliser supply was used as raw material for VO fertiliser production, and also as 15N-labelled sheep feed to obtain 15N-labelled manure. The labelled faeces fraction was used as raw material for the AO fertiliser. The VO fertiliser was obtained after an acidic and an enzyme-driven hydrolysis. The AO fertiliser was obtained after acidic hydrolysis. The VO liquid fertiliser presented an N concentration of 330 mg·L-1, 85% of total N was organic, while ammonium and nitrate N accounted for 55% and 45% of the mineral nitrogen fraction, respectively. This fertiliser also exhibited high K, Ca and S concentrations and notable values for the remaining macro- and micronutrients. The AO liquid fertiliser had a similar total N concentration (496 mg·L-1, 82% of total N in an organic form) to that of VO, but its mineral N fraction significantly differed, which came in a predominantly (95%) ammonia form. It also had a high content of N, P, K and other macronutrients, and sufficient Fe, Zn, Mn, Cu and B levels, which suggests its suitability as a potential fertiliser. The percentage of 15N enrichment in both VO and AO liquid fertilisers exceeded 2% 15N atom excess, which enabled their use in subsequent assays run to assess nitrogen uptake efficiency. PMID:26982183

  7. Non-homogeneity of isotopic labelling in 15N gas flux studies: theory, some observations and possible lessons

    NASA Astrophysics Data System (ADS)

    Well, Reinhard; Buchen, Caroline; Deppe, Marianna; Eschenbach, Wolfram; Gattinger, Andreas; Giesemann, Anette; Krause, Hans-Martin; Lewicka-Szczebak, Dominika

    2015-04-01

    addressing cases iii. and iv.. Furthermore we present some experimental data illustrating this. These include two data sets from denitrification experiments exhibiting substantial deviations in 15N enrichment between the N pools producing N2 and N2O. Moreover, results from a lab incubation study to quantify NH4+-derived N2O with increasing NH4+ amendment under conditions favouring nitrification are shown, were non-labelled NH4+ was added together with 15N labelled NO3-. Here we found large deviations between the 15N enrichment of NO3- in extracted soil water and the 15N enrichment of the labelled N pool as calculated from N2O isotopologues (Bergsma et al., 2001). We think that this reflects type iv. bias, probably because enrichment of NO3- in anoxic micro-sites was less diluted by non-labelled NO3- from nitrification compared to NO3- in oxic zones. Our data analysis provides a means to overcome bias iv. and thus to obtain correct source apportionment. References: Arah, J.R.M. (1992): Soil Sci. Soc. Am. J. 56, 795 - 800, 1992. Bergsma, T. et al. (2001): Env. Sci. & Technol. 35(21): 4307-4312. Hauck, R.D., et al.(1958): Soil Science 86, 287 - 291, 1958. Lewicka-Szczebak, D. et al.(2013): Rapid Comm. Mass Spectrom., 27 1548-1558. Müller, C. et al. (2004): Soil Biol. Biochem. 36(4): 619-632. Mulvaney, R.L.(1984):. Soil Sci. Soc. Am. J. 48:690 - 692. Spott, O, et al.. (2006): Rapid Comm. Mass Spectrom., 20: 3267-3274. Spott, O. and C. F. Stange (2007): Rapid Comm. Mass Spectrom., 21: 2398-2406.

  8. Refining cotton-wick method for 15N plant labelling.

    NASA Astrophysics Data System (ADS)

    Fustec, Joëlle; Mahieu, Stéphanie

    2010-05-01

    The symbiosis Fabaceae/Rhizobiaceae plays a critical role in the nitrogen cycle. It gives the plant the ability to fix high amounts of atmospheric N. A part of this N can be transferred to the soil via rhizodeposition. The contribution of Fabaceae to the soil N pool is difficult to measure, since it is necessary for assessing N benefits for other crops, for soil biological activity, and for reducing water pollution in sustainable agriculture (Fustec, 2009). The aim of this study was to test and improve the reliability of the 15N cotton-wick method for measuring the soil N derived from plant rhizodeposition (Mahieu et al., 2007). The effects of the concentration of the 15N-urea labelling solution and of the feeding frequency (continuous or pulses) on the assessment of nitrogen rhizodeposition were studied in two greenhouse experiments using the field pea (Pisum sativum L.) and the non-nodulating isoline P2. The plant parts and the soil were prepared for 15N:14N measurements for assessing N rhizodeposition (Mahieu et al., 2009). The fraction of plants' belowground nitrogen allocated to rhizodeposition in both Frisson pea and P2 was 20 to more than 50% higher when plants were labelled continuously than when they were labelled using fortnightly pulses. Our results suggested that when 15N root enrichment was high, nitrogen rhizodeposition was underestimated only for plants that were 15N-fed by fortnightly pulses, and not in plants 15N-fed continuously. This phenomenon was especially observed for plants relying on symbiotic N fixation for N acquisition; it may be linked to the concentration of the labelling solution. In conclusion, N rhizodeposition assessment was strongly influenced by the 15N-feeding frequency and the concentration of the labelling solution. The estimation of N rhizodeposition was more reliable when plants were labelled continuously with a dilute solution of 15N urea. Fustec et al. 2009. Agron. Sustain. Dev., DOI 10.1051/agro/2009003, in press. Mahieu

  9. Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins

    SciTech Connect

    McIntosh, L.P.; Griffey, R.H.; Muchmore, D.C.; Nielson, C.P.; Redfield, A.G.; Dahlquist, F.W.

    1987-03-01

    A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on /sup 15/N (or /sup 13/C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-/sup 15/N (or /sup 13/C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from /sup 15/N-bonded protons in the backbone and sidechains of uniformly /sup 15/N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-/sup 15/N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of /sup 15/N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple /sup 15/N and /sup 13/C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl(/sup 15/N)alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

  10. Fate of orally administered 15N-labeled polyamines in rats bearing solid tumors.

    PubMed

    Kobayashi, Masaki; Xu, Yong Ji; Samejima, Keijiro; Goda, Hitomi; Niitsu, Masaru; Takahashi, Masakazu; Hashimoto, Yoshiyuki

    2003-03-01

    We studied absorption, distribution, metabolism, and excretion of polyamines (putrescine, spermidine, and spermine) in the gastrointestinal tract using (15)N-labeled polyamines as tracers and ionspray ionization mass spectrometry (IS-MS). The relatively simple protocol using rats bearing solid tumors provided useful information. Three (15)N-labeled polyamines that were simultaneously administered were absorbed equally from gastrointestinal tract, and distributed within tissues at various concentrations. The uptake of (15)N-spermidine seemed preferential to that of (15)N-spermine since the concentrations of (15)N-spermidine in the liver and tumors were higher, whereas those of (15)N-spermine were higher in the kidney, probably due to the excretion of excess extracellular spermine. Most of the absorbed (15)N-putrescine seemed to be lost, suggesting blood and tissue diamine oxidase degradation. Concentrations of (15)N-spermidine and (15)N-spermine in the tumor were low. We also describe the findings from two rats that were administered with (15)N-spermine. The tissue concentrations of (15)N-spermine were unusually high, and significant levels of (15)N-spermidine were derived from (15)N-spermine in these animals.

  11. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    PubMed Central

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  12. In vivo, large-scale preparation of uniformly (15)N- and site-specifically (13)C-labeled homogeneous, recombinant RNA for NMR studies.

    PubMed

    Le, My T; Brown, Rachel E; Simon, Anne E; Dayie, T Kwaku

    2015-01-01

    Knowledge of how ribonucleic acid (RNA) structures fold to form intricate, three-dimensional structures has provided fundamental insights into understanding the biological functions of RNA. Nuclear magnetic resonance (NMR) spectroscopy is a particularly useful high-resolution technique to investigate the dynamic structure of RNA. Effective study of RNA by NMR requires enrichment with isotopes of (13)C or (15)N or both. Here, we present a method to produce milligram quantities of uniformly (15)N- and site-specifically (13)C-labeled RNAs using wild-type K12 and mutant tktA Escherichia coli in combination with a tRNA-scaffold approach. The method includes a double selection protocol to obtain an E. coli clone with consistently high expression of the recombinant tRNA-scaffold. We also present protocols for the purification of the tRNA-scaffold from a total cellular RNA extract and the excision of the RNA of interest from the tRNA-scaffold using DNAzymes. Finally, we showcase NMR applications to demonstrate the benefit of using in vivo site-specifically (13)C-labeled RNA. © 2015 Elsevier Inc. All rights reserved.

  13. NMR studies of the stability, protonation States, and tautomerism of (13)C- AND (15)N-labeled aldimines of the coenzyme pyridoxal 5'-phosphate in water.

    PubMed

    Chan-Huot, Monique; Sharif, Shasad; Tolstoy, Peter M; Toney, Michael D; Limbach, Hans-Heinrich

    2010-12-28

    We have measured the pH-dependent (1)H, (13)C, and (15)N NMR spectra of pyridoxal 5'-phosphate ((13)C(2)-PLP) mixed with equal amounts of either doubly (15)N-labeled diaminopropane, (15)N(α)-labeled l-lysine, or (15)N(ε)-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their (13)C and (15)N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK(a) values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.

  14. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    SciTech Connect

    Jones, R.A.

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  15. The Cyanide Ligands of [FeFe] Hydrogenase: Pulse EPR Studies of 13C and 15N-Labeled H-Cluster

    PubMed Central

    2015-01-01

    The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic 13C hyperfine interactions for the two CN– ligands—a reporter of spin density on their respective coordinating iron ions—collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [15N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine. PMID:25133957

  16. Elucidating mineralisation-immobilisation dynamics in a grassland soil using triple 15N labelling in the field combined with a 15N tracing laboratory approach

    NASA Astrophysics Data System (ADS)

    Kleineidam, Kristina; Müller, Christoph

    2017-04-01

    Mineralisation is a key N transformation process supplying reactive nitrogen (N) to terrestrial ecosystems. The various soil organic matter fractions contribute to the total mineralisation according to their turnover characteristic. However, the exact mechanism and the gross dynamics of the various processes are not well understood. In this study we investigated the mineralisation-immobilisation dynamics in a grassland soil by a combined field-laboratory study. Eighteen microplots were established at a field site receiving 50 kg N ha-1 as ammonium nitrate. In nine (3 x 3) respective plots the ammonium, or the nitrate, or both moieties were 15N labelled at 60 atom%. Previous studies with this soil showed that rapid turnover occurred and available N would partly be immobilised by the microbial biomass increasing the 15N label of the soil organic nitrogen pool in the field. After one year, soil samples were taken from the 15N treated and the so far non-labelled plots and examined in a laboratory study (for details of the setup see: Müller et al., 2004). While the previously differentially 15N labelled field soils were now supplied with unlabelled ammonium nitrate, the previously unlabelled soils were now treated with either 15N labelled ammonium nitrate similar to the 15N treatments established in the field, resulting in six different 15N treatments in total. The incubation study was carried out over a two week period and data were analysed with the Ntrace model to quantify the simultaneously occurring gross N transformations while optimizing a single parameter set for all six treatments. Thus, the appearance of 15N from the previously labelled soils and the dilution of the 15N in the recently labelled treatments were assumed to be driven by the same processes and activities and were used to constrain the 15N tracing model. This approach allowed us to estimate the individual gross N transformation rates with a much higher accuracy than if only a common triple

  17. Affordable uniform isotope labeling with (2)H, (13)C and (15)N in insect cells.

    PubMed

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D

    2015-06-01

    For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80% can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.

  18. Production of 15N-labeled α-amanitin in Galerina marginata

    PubMed Central

    DuBois, Brandon; Sgambelluri, R. Michael; Angelos, Evan R.; Li, Xuan; Holmes, Daniel

    2015-01-01

    α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes directly from A. phalloides, the death cap mushroom, which is collected from its natural habitat. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprobic mushroom that grows and produces α-amanitin in culture, we describe a method for producing 15N-labeled α-amanitin using growth media containing 15N as sole nitrogen source. A key to success was preparing 15N-enriched yeast extract via a novel method designated “glass bead-assisted maturation.” In the presence of the labeled yeast extract and 15N-NH4Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR. PMID:26100667

  19. Production of (15)N-labeled α-amanitin in Galerina marginata.

    PubMed

    Luo, Hong; DuBois, Brandon; Sgambelluri, R Michael; Angelos, Evan R; Li, Xuan; Holmes, Daniel; Walton, Jonathan D

    2015-09-01

    α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces α-amanitin in culture, we describe a method for producing (15)N-labeled α-amanitin using growth media containing (15)N as sole nitrogen source. A key to success was preparing (15)N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and (15)N-NH4Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR.

  20. The “Speedy” Synthesis of Atom-Specific 15N Imino/Amido-Labeled RNA

    PubMed Central

    Kreutz, Christoph; Micura, Ronald

    2016-01-01

    Although numerous reports on the synthesis of atom-specific 15N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of 15N(1) adenosine and 15N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve 15N(3) uridine and 15N(3) cytidine amidites in order to tap full potential of 1H/15N/15N-COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered. PMID:26237536

  1. Preparation of 13C/15N-labeled oligomers using the polymerase chain reaction

    DOEpatents

    Chen, Xian; Gupta, Goutam; Bradbury, E. Morton

    2001-01-01

    Preparation of .sup.13 C/.sup.15 N-labeled DNA oligomers using the polymerase chain reaction (PCR). A PCR based method for uniform (.sup.13 C/.sup.15 N)-labeling of DNA duplexes is described. Multiple copies of a blunt-ended duplex are cloned into a plasmid, each copy containing the sequence of interest and restriction Hinc II sequences at both the 5' and 3' ends. PCR using bi-directional primers and uniformly .sup.13 C/.sup.15 N-labeled dNTP precursors generates labeled DNA duplexes containing multiple copies of the sequence of interest. Twenty-four cycles of PCR, followed by restriction and purification, gave the uniformly .sup.13 C/.sup.15 N-labeled duplex sequence with a 30% yield. Such labeled duplexes find significant applications in multinuclear magnetic resonance spectroscopy.

  2. Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples.

    PubMed

    Truhlar, Stephanie M E; Cervantes, Carla F; Torpey, Justin W; Kjaergaard, Magnus; Komives, Elizabeth A

    2008-09-01

    Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.

  3. NMR studies on /sup 15/N-labeled creatine (CR), creatinine (CRN), phosphocreatine (PCR), and phosphocreatinine (PCRN), and on barriers to rotation in creatine kinase-bound creatine in the enzymatic reaction

    SciTech Connect

    Kenyon, G.L.; Reddick, R.E.

    1986-05-01

    Recently, the authors have synthesized /sup 15/N-2-Cr, /sup 15/N-3-Crn, /sup 15/N-2-Crn, /sup 15/N-3-PCrn, /sup 15/N-3-PCr, and /sup 15/N-2-PCr. /sup 1/H, /sup 15/N, /sup 31/P NMR data show that Crn protonates exclusively at the non-methylated ring nitrogen, confirm that PCrn is phosphorylated at the exocyclic nitrogen, and demonstrate that the /sup 31/P-/sup 15/N one-bond coupling constant in /sup 15/N-3-PCr is 18 Hz, not 3 Hz as previously reported by Brindle, K.M., Porteous, R. and Radda, G.K.. The authors have found that creatine kinase is capable of catalyzing the /sup 14/N//sup 15/N positional isotope exchange of 3-/sup 15/N-PCr in the presence of MgADP, but not in its absence. Further, the exchange does not take place when labeled PCr is resynthesized exclusively from the ternary complex E X Cr X MgATP as opposed to either E X Cr or free Cr. This suggests that the enzyme both imparts an additional rotational barrier to creatine in the complex and catalyzes the transfer of phosphoryl group with essentially complete regiospecificity.

  4. NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris.

    PubMed

    de Lamotte, F; Boze, H; Blanchard, C; Klein, C; Moulin, G; Gautier, M F; Delsuc, M A

    2001-07-01

    Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native (15)N-labeled protein. Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted. Control of all nitrogen sources permits very high labeling yields. As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification. Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase. The detection limit for a (15)N-labeled protein is estimated as 20 microM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor. This concentration is reached by most reported preparations in P. pastoris. Further concentration by ultrafiltration would compensate for lower production. This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method. Copyright 2001 Academic Press.

  5. Nitrogen turnover of three different agricultural soils determined by 15N triple labelling

    NASA Astrophysics Data System (ADS)

    Fiedler, Sebastian R.; Kleineidam, Kristina; Strasilla, Nicol; Schlüter, Steffen; Reent Köster, Jan; Well, Reinhard; Müller, Christoph; Wrage-Mönnig, Nicole

    2017-04-01

    To meet the demand for data to improve existing N turnover models and to evaluate the effect of different soil physical properties on gross nitrogen (N) transformation rates, we investigated two arable soils and a grassland soil after addition of ammonium nitrate (NH4NO3), where either ammonium (NH4+), or nitrate (NO3-), or both pools have been labelled with 15N at 60 atom% excess (triple 15N tracing method). Besides NH4+, NO3- and nitrite (NO2-) contents with their respective 15N enrichment, nitrous oxide (N2O) and dinitrogen (N2) fluxes have been determined. Each soil was adjusted to 60 % of maximum water holding capacity and pre-incubated at 20˚ C for two weeks. After application of the differently labelled N fertilizer, the soils were further incubated at 20˚ C under aerobic conditions in a He-N2-O2 atmosphere (21 % O2, 76 He, 2% N2) to increase the sensitivity of N2 rates via the 15N gas flux method. Over a 2 week period soil N pools were quantified by 2 M KCl extraction (adjusted to pH 7 to prevent nitrite losses) (Stevens and Laughlin, 1995) and N gas fluxes were measured by gas chromatography in combination with IRMS. Here, we present the pool sizes and fluxes as well as the 15N enrichments during the study. Results are discussed in light of the soil differences that were responsible for the difference in gross N dynamics quantified by the 15N tracing model Ntrace (Müller et al., 2007). References Müller, C., T. Rütting, J. Kattge, R.J. Laughlin, and R.J. Stevens, (2007) Estimation of parameters in complex 15N tracing models by Monte Carlo sampling. Soil Biology & Biochemistry. 39(3): p. 715-726. Stevens, R.J. and R.J. Laughlin, (1995) Nitrite transformations during soil extraction with potassium chloride. Soil Science Society of America Journal. 59(3): p. 933-938.

  6. In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics.

    PubMed

    Ippel, Johannes H; Pouvreau, Laurice; Kroef, Toos; Gruppen, Harry; Versteeg, Geurt; van den Putten, Peter; Struik, Paul C; van Mierlo, Carlo P M

    2004-01-01

    Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.

  7. Syntheses of all singly labeled [15N]adenines: Mass spectral fragmentation of adenine

    PubMed Central

    Barrio, Maria Del Carmen G.; Scopes, David I. C.; Holtwick, Joseph B.; Leonard, Nelson J.

    1981-01-01

    Syntheses of all five of the singly labeled [15N]adenines are now provided. The presence or absence of two-bond 15N-1H spin couplings in their 1H NMR spectra confirm the location of the isotope in each case. The fragmentation patterns in their mass spectra are indicative of the sequential losses of HCN units and of CH2N2 from adenine upon electron impact. PMID:16593042

  8. Decomposition and nitrogen dynamics of 15N-labeled leaf, root, and twig litter in temperate coniferous forests

    Treesearch

    T.L. van Huysen; M.E. Harmon; S.S. Perakis; H. Chen

    2013-01-01

    Litter nutrient dynamics contribute significantly to biogeochemical cycling in forest ecosystems. We examined how site environment and initial substrate quality influence decomposition and nitrogen (N) dynamics of multiple litter types. A 2.5-year decomposition study was installed in the Oregon Coast Range and West Cascades using 15N-labeled...

  9. Overhauser DNP with 15N labelled Frémy's salt at 0.35 Tesla.

    PubMed

    Türke, Maria-Teresa; Parigi, Giacomo; Luchinat, Claudio; Bennati, Marina

    2012-01-14

    The effectiveness of dynamic nuclear polarization (DNP) as a tool to enhance the sensitivity of liquid state NMR critically depends on the choice of the optimal polarizer molecule. In this study the performance of (15)N labelled Frémy's salt as a polarizing agent in Overhauser DNP is investigated in detail at X-band (0.35 T, 9.7 GHz EPR, 15 MHz (1)H NMR) and compared to that of TEMPONE-D,(15)N employed in previous studies. Both radicals provide similar maximum enhancements of the solvent water protons under similar conditions but a different saturation behaviour. The factors determining the enhancement and effective saturation were measured independently by EPR, ELDOR and NMRD and are shown to fulfil the Overhauser equation. In particular, following the theory of EPR saturation we provide analytical solutions for the dependence of the enhancement on the microwave field strength in terms of saturation transfer between two coupled hyperfine lines undergoing spin exchange. The negative charge of the radical in Frémy's salt solutions can explain the peculiar properties of this polarizing agent and indicates different suitable application areas for the two types of nitroxide radicals.

  10. Nirtogen-15-labeled oligodeoxynucleotides. 4. Tetraplex formation of d[G({sup 15}N{sup 7})GTTTTTGG] and d[T({sup 15}N{sup 7})GGGT] monitored by {sup 1}H detected {sup 15}N NMR

    SciTech Connect

    Gaffney, B.L.; Chuan Wang; Jones, R.A.

    1992-05-20

    The authors have synthesized two molecules containing [7-{sup 15}N]-labeled 2{prime}-deoxyguanosine, d[G({sup 15}N{sup 7})GTTTTTGG], and d[T({sup 15}N{sup 7})GGGT] which, under appropriate conditions, will form tetramolecular complexes. The {sup 15}N chemical shifts of these molecules and of their Watson-Crick duplexes, d[G({sup 15}N{sup 7})GTTTTTGG]-d[CCAAAAACC] and d[T({sup 15}N{sup 7})GGGT]-d[ACCCA], were monitored as a function of temperature. The {sup 15}N chemical shift of the labeled N7 atom in each tetramolecular complex shows a similar temperature dependence, and the chemical shifts are not signal-averaged. The similarity of the chemical shifts for the tetraplex and single strand structures, and the difference seen for the two duplexes, are consistent with the different degrees of hydrogen bonding to the N7 which could be expected in each case. Thus, although more examples will be required to establish the generality of these observations, a purine [7-{sup 15}N] label appears to be able to monitor groove interactions, including hydration. 28 refs., 6 figs., 5 tabs.

  11. Nitrogen mineralization from selected /sup 15/N-labelled crop residues and humus as affected by inorganic nitrogen

    SciTech Connect

    Santos, J.A.

    1987-01-01

    The use of cover crops or crop residues as a source of N to succeeding crops has become a matter of increasing importance for economic and environmental reason. Greenhouse and field studies were conducted to determine the N contribution of four /sup 15/N labelled crop residues, rye (Secale cereale L.), wheat (Triticum aestivum L.), crimson clover (Trifolium encarnatum L.), and hairy vetch (Vicia sativa L.), to successive crops and to evaluate the effect of different organic (ON) and inorganic N (IN) combinations on mineralization of the above residues. Total /sup 15/N recovery from the residues ranged from 51% to 85% and 4% to 74% for the greenhouse and field studies, respectively.

  12. NMR studies of active-site properties of human carbonic anhydrase II by using (15) N-labeled 4-methylimidazole as a local probe and histidine hydrogen-bond correlations.

    PubMed

    Shenderovich, Ilya G; Lesnichin, Stepan B; Tu, Chingkuang; Silverman, David N; Tolstoy, Peter M; Denisov, Gleb S; Limbach, Hans-Heinrich

    2015-02-09

    By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

    PubMed Central

    Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M. Francesca

    2014-01-01

    Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or 2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components

  14. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant isotope labeling.

    PubMed

    Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M Francesca

    2014-01-16

    Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as (13)C with (15)N, (18)O or (2)H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation(1-4). From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage(5-7). The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing (13)C and (15)N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous (13)C and (15)N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%(13)C and 6.7 atom%(15)N uniform plant label, or material that is differentially labeled by up to 1.29 atom%(13)C and 0.56 atom%(15)N in its metabolic and structural components (hot water extractable and hot water

  15. Exploring the nitrogen ingestion of aphids--a new method using electrical penetration graph and (15)N labelling.

    PubMed

    Kuhlmann, Franziska; Opitz, Sebastian E W; Inselsbacher, Erich; Ganeteg, Ulrika; Näsholm, Torgny; Ninkovic, Velemir

    2013-01-01

    Studying plant-aphid interactions is challenging as aphid feeding is a complex process hidden in the plant tissue. Here we propose a combination of two well established methods to study nutrient acquisition by aphids focusing on the uptake of isotopically labelled nitrogen ((15)N). We combined the Electrical Penetration Graph (EPG) technique that allows detailed recording of aphid feeding behaviour and stable isotope ratio mass spectrometry (IRMS) to precisely measure the uptake of nitrogen. Bird cherry-oat aphids Rhopalosiphum padi L. (Hemiptera, Aphididae) fed for 24 h on barley plants (Hordeum vulgare L., cultivar Lina, Poaceae) that were cultivated with a (15)N enriched nutrient solution. The time aphids fed in the phloem was strongly positive correlated with their (15)N uptake. All other single behavioural phases were not correlated with (15)N enrichment in the aphids, which corroborates their classification as non-feeding EPG phases. In addition, phloem-feeding and (15)N enrichment of aphids was divided into two groups. One group spent only short time in the phloem phase and was unsuccessful in nitrogen acquisition, while the other group displayed longer phloem-feeding phases and was successful in nitrogen acquisition. This suggests that several factors such as the right feeding site, time span of feeding and individual conditions play a role for the aphids to acquire nutrients successfully. The power of this combination of methods for studying plant-aphid interactions is discussed.

  16. Exploring the Nitrogen Ingestion of Aphids — A New Method Using Electrical Penetration Graph and 15N Labelling

    PubMed Central

    Kuhlmann, Franziska; Opitz, Sebastian E. W.; Inselsbacher, Erich; Ganeteg, Ulrika; Näsholm, Torgny; Ninkovic, Velemir

    2013-01-01

    Studying plant-aphid interactions is challenging as aphid feeding is a complex process hidden in the plant tissue. Here we propose a combination of two well established methods to study nutrient acquisition by aphids focusing on the uptake of isotopically labelled nitrogen (15N). We combined the Electrical Penetration Graph (EPG) technique that allows detailed recording of aphid feeding behaviour and stable isotope ratio mass spectrometry (IRMS) to precisely measure the uptake of nitrogen. Bird cherry-oat aphids Rhopalosiphum padi L. (Hemiptera, Aphididae) fed for 24 h on barley plants (Hordeum vulgare L., cultivar Lina, Poaceae) that were cultivated with a 15N enriched nutrient solution. The time aphids fed in the phloem was strongly positive correlated with their 15N uptake. All other single behavioural phases were not correlated with 15N enrichment in the aphids, which corroborates their classification as non-feeding EPG phases. In addition, phloem-feeding and 15N enrichment of aphids was divided into two groups. One group spent only short time in the phloem phase and was unsuccessful in nitrogen acquisition, while the other group displayed longer phloem-feeding phases and was successful in nitrogen acquisition. This suggests that several factors such as the right feeding site, time span of feeding and individual conditions play a role for the aphids to acquire nutrients successfully. The power of this combination of methods for studying plant-aphid interactions is discussed. PMID:24376642

  17. Soil nutrient cycling in reclamation and natural boreal forest soils using 15N labeled aspen leaf litter

    NASA Astrophysics Data System (ADS)

    Norris, Charlotte; Quideau, Sylvie; Landhäusser, Simon

    2013-04-01

    A region of the boreal forest, located north of Fort McMurray, Alberta, is currently being disturbed by oil sands mining. Conditional to the allowance of mining is the requirement that the land be returned to an equivalent land capability. One method to determine if the equivalent capability has been met is to know if the reconstructed sites are self sustaining in terms of central ecosystem processes such as nutrient cycling. This study set out to compare nutrient cycling among the litter and live vegetation on reconstructed, harvested and undisturbed forest sites. All sites were dominated by an aspen tree canopy (Populus tremuloides Michx.). Nutrient cycling was monitored through the addition of 15N labeled aspen leaf litter to the forest floor over four sampling periods (0, 4, 12 and 16 months) and testing key soil response variables such as nutrient supply, microbial community, and organic matter composition. Over the entire collection period the soil microbial biomass for harvested and reconstructed soils was similar in quantity while the undisturbed forest soil was always three times greater in biomass. With the addition of 15N labeled leaves, the δ15N of microbial biomass increased across the incubation for all sites and the harvested site showed the greatest response. Furthermore the increased concentration of 15N in the live vegetation indicates that nutrient cycling was occurring on all sites.

  18. Synthesis and biosynthesis of {sup 13}C-, {sup 15}N-labeled deoxynucleosides useful for biomolecular structural determinations

    SciTech Connect

    Ashburn, D.A.; Garcia, K.; Hanners, J.L.; Silks, L.A. III; Unkefer, C.J.

    1994-12-01

    Currently, there is a great emphasis on elucidating the structure, function, and dynamics of DNA. Much of the research involved in this study uses nuclear magnetic resonance (NMR) spectroscopy. Effective use of NMR spectroscopy for DNA molecules with mw > 10,000 requires stable isotope enrichment. We present strategies for site-specific isotopic labeling of the purine bases adenosine and guanosine and the biosynthesis of (U-{sup 13}C, {sup 15}N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2{prime}-deoxy-(amino-{sup 15}N)adenosine (dA). The resulting d(amino-{sup 15}N)A is used in a series of reactions to synthesize 2{prime}-deoxy-(2-{sup 13}C,1,amino-{sup 15}N{sub 2})guanosine or any combination thereof. An improved biosynthesis of labeled DNA has been accomplished using Methylobacterium extorquens AS1. Each liter of growth medium contains 4 g of methanol to yield 1 g of lyophilized cells. As much as 200 mg of RNA per liter of culture has been obtained. We are currently developing large-scale isolation protocols. General synthetic pathways to oligomeric DNA will be presented.

  19. A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

    PubMed

    Nicolás Carcelén, Jesús; Marchante-Gayón, Juan Manuel; González, Pablo Rodríguez; Valledor, Luis; Cañal, María Jesús; Alonso, José Ignacio García

    2017-08-18

    The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, (15)N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing (15)NH4Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the (15)N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only

  20. Nitrogen removal in maturation ponds: tracer experiments with 15N-labelled ammonia.

    PubMed

    Camargo Valero, M A; Mara, D D

    2007-01-01

    A primary maturation pond (M1) was spiked with labelled ammonium chloride (15NH4Cl) to track ammonium transformations associated with algal uptake and subsequent sedimentation. Conventional sampling based on grab samples collected from M1 influent, water column and effluent, and processed for unfiltered and filtered TKN, ammonium, nitrite and nitrate, found low total nitrogen removal (8%) and high ammonium nitrogen removal (90%). Stable isotope analysis of 15N from suspended organic and ammonium nitrogen fractions in M1 effluent revealed that labelled ammonium was mainly found in the organic fraction (69% of the 15N recovered), rather than the inorganic fraction (5%). Algal uptake was the predominant pathway for ammonia removal, even though conditions were favourable for ammonia volatilization (8.9 < pH <10.1 units, 15.2 < temperature <18.8 degrees C). Total nitrogen was removed by ammonia volatilization at 15 g N/ha d (3%), organic nitrogen sedimentation at 105 g N/ha d (20%), and in-pond accumulation due to algal uptake at 377 g N/ha d (71%). Algal uptake of ammonium and subsequent sedimentation and retention in the benthic sludge, after partial ammonification of the algal organic nitrogen, is thus likely to be the dominant mechanism for permanent nitrogen removal in maturation ponds during warm summer months in England.

  1. Stereospecific assignments of glycine in proteins by stereospecific deuteration and {sup 15}N labeling

    SciTech Connect

    Hansen, A.P.; Curley, R.W. Jr.; Panigot, M.J.; Fesik, S.W.

    1994-12-01

    Stereospecific assignments are important for accurately determining the three-dimensional structures of proteins through the use of multidimensional NMR techniques. It is especially important to stereospecifically assign the glycine {alpha}-protons in proteins because of the potential for different backbone conformations of this residue. These stereospecific assignments are critical for interpreting the {sup 3}J{sub NH,{alpha}H} coupling constants and NOEs involving the glycine {alpha}-protons that determine the conformation of this part of the protein. However, it is often difficult to unambiguously obtain the stereospecific assignments for glycine residues by using only NOE data. In this poster, we present a method for unambiguous, stereospecific assignment of the {alpha}-protons of glycine residues. This method involves synthesis of stereo-specifically deuterated and {sup 15}N-labeled Gly using a slightly modified procedure originally described by Woodard and coworkers for the stereoselective deuteration of glycine. The stereospecifically deuterated and {sup 15}N-labeled Gy has been incorporated into recombinant proteins expressed in both bacterial systems (FKBP) and mammalian cells (u-PA). Two- and three-dimensional isotope-filtered and isotope-edited NMR experiments were used to obtain the stereospecific assignments of the glycine {alpha}-protons for these proteins.

  2. Reactions, characterization and uptake of ammoxidized kraft lignin labeled with 15N.

    PubMed

    Ramírez, F; Varela, G; Delgado, E; López-Dellamary, F; Zúñiga, V; González, V; Faix, O; Meier, D

    2007-05-01

    Ammoxidation of kraft lignin was carried out in a Parr reactor using (15)NH(3) as the main nitrogen source. Reaction parameters were set up until a total nitrogen content of approximately 13 wt.% in lignin was achieved, in accordance with conditions of previous studies. Analytical tools such as FTIR, Py-GC/MS, and solid state NMR were used in this research. The nature of nitrogen bondings is discussed. The incorporation of the (15)N from ammoxidized lignin was followed in pumpkins (Zucchini cucurbita pepo L.) by means of (15)N emission spectroscopy.

  3. /sup 18/O isotope shift in /sup 15/N NMR spectroscopy. 2. Synthesis of /sup 15/N, /sup 18/O-labeled hydroxylamine hydrochloride

    SciTech Connect

    Rajendran, G.; Van Etten, R.L.

    1986-03-12

    Since hydroxylamine can serve as a key intermediate in the synthesis of a variety of compounds, the synthesis of (/sup 15/N, /sup 18/O)-labelled hydroxylamine hydrochloride was undertaken. Published procedures for the synthesis of hydroxylamine resulted in poor yields in some cases and in lower percentage of /sup 18/O in the product than expected in other cases. The compound was synthesized in dry tetrahydrofuran (THF) by treating NaNO/sub 2/ with borane-methyl sulfide. The course of the reaction was examined using /sup 11/B NMR spectroscopy, and the product yield was 74%. The /sup 18/O enrichment was demonstrated by both mass spectrometry and /sup 15/N NMR of the isolated acetoxime. 23 references, 1 figure.

  4. A facile method for expression and purification of 15N isotope-labeled human Alzheimer's β-amyloid peptides from E. coli for NMR-based structural analysis

    PubMed Central

    Armand, Tara; Ball, K. Aurelia; Chen, Anna; Pelton, Jeffrey G.; Wemmer, David E.; Head-Gordon, Teresa

    2016-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality 15N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with 15N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the 15N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure 15N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼6 mg/L culture for 15N isotope-labeled Aβ42 peptide. Mass spectrometry and 1H–15N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with 15N and 13C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants. PMID:26231074

  5. Tracking the incorporation of 15N from labeled beech litter into mineral-organic associations

    NASA Astrophysics Data System (ADS)

    Kleber, M.; Hatton, P.; Derrien, D.; Lajtha, K.; Zeller, B.

    2008-12-01

    Nitrogen containing organic compounds are thought to have a role in the complex web of processes that control the turnover time of soil organic matter. The sequential density fractionation technique is increasingly used for the purpose of investigating the association of organic materials with the mineral matrix. Organic materials in the denser fractions (>2.0 kg L-1) typically show 13C NMR signals indicative of carbohydrate and aliphatic structures, an absence of lignin and tannin structures and a narrow C:N ratio, suggesting a microbial origin of organic matter in these fractions. Here we take advantage of a labeling experiment conducted at two different sites in Germany and in France to investigate the incorporation of organic nitrogen into physical fractions of increasing density, representing a proximity gradient to mineral surfaces. 15N labeled beech litter was applied to two acidic forest topsoils 8 and 12 years ago. Although there are differences in the distribution patterns between the two soils, and the majority of the organic nitrogen was recovered in fractions representing organic matter of plant origin and not bound to the mineral matrix, our data clearly show that after a decade, significant amounts of the nitrogen had been incorporated in mineral-organic fractions of supposedly slow turnover. It remains to be shown to which extent the N in the densest fractions was incorporated by soil microbiota and associated with mineral surfaces in organic form or adsorbed to mineral surfaces in inorganic form (NH4+).

  6. Nitrogen removal in Myriophyllum aquaticum wetland microcosms for swine wastewater treatment: (15) N-labelled nitrogen mass balance analysis.

    PubMed

    Zhang, Shunan; Liu, Feng; Xiao, Runlin; He, Yang; Wu, Jinshui

    2017-01-01

    Ecological treatments are effective for treating agricultural wastewater. In this study, wetland microcosms vegetated with Myriophyllum aquaticum were designed for nitrogen (N) removal from two strengths of swine wastewater, and (15) N-labelled ammonium (NH4(+) -N) was added to evaluate the dominant NH4(+) -N removal pathway. The results showed that 98.8% of NH4(+) -N and 88.3% of TN (TN: 248.6 mg L(-1) ) were removed from low-strength swine wastewater (SW1) after an incubation of 21 days, with corresponding values for high-strength swine wastewater (SW2) being 99.2% of NH4(+) -N and 87.8% of TN (TN: 494.9 mg L(-1) ). Plant uptake and soil adsorption respectively accounted for 24.0% and 15.6% of the added (15) N. Meanwhile, above-ground tissues of M. aquaticum had significantly higher biomass and TN content than below-ground (P < 0.05). (15) N mass balance analysis indicated that gas losses contributed 52.0% to the added (15) N, but the N2 O flux constituted only 7.5% of total gas losses. The dynamics of NO3(-) -N and N2 O flux revealed that strong nitrification and denitrification occurred in M. aquaticum microcosms, which was a dominant N removal pathway. These findings demonstrated that M. aquaticum could feasibly be used to construct wetlands for high N-loaded animal wastewater treatment. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  7. Determination of Multimodal Isotopic Distributions: The Case of a (15)N Labeled Protein Produced into Hairy Roots.

    PubMed

    Trouillard, Romain; Hubert-Roux, Marie; Tognetti, Vincent; Guilhaudis, Laure; Plasson, Carole; Menu-Bouaouiche, Laurence; Coquet, Laurent; Guerineau, François; Hardouin, Julie; Ele Ekouna, Jean-Pierre; Cosette, Pascal; Lerouge, Patrice; Boitel-Conti, Michèle; Afonso, Carlos; Ségalas-Milazzo, Isabelle

    2015-06-16

    Isotopic labeling is widely used in various fields like proteomics, metabolomics, fluxomics, as well as in NMR structural studies, but it requires an efficient determination of the isotopic enrichment. Mass spectrometry is the method of choice for such analysis. However, when complex expression systems like hairy roots are used for production, multiple populations of labeled proteins may be obtained. If the isotopic incorporation determination is actually well-known for unimodal distributions, the multimodal distributions have scarcely been investigated. Actually, only a few approaches allow the determination of the different labeled population proportions from multimodal distributions. Furthermore, they cannot be used when the number of the populations and their respective isotope ratios are unknown. The present study implements a new strategy to measure the (15)N labeled populations inside a multimodal distribution knowing only the peptide sequence and peak intensities from mass spectrometry analyses. Noteworthy, it could be applied to other elements, like carbon and hydrogen, and extended to a larger range of biomolecules.

  8. Partitioning Residue-derived and Residue-induced Emissions of N2O Using 15N-labelled Crop Residues

    NASA Astrophysics Data System (ADS)

    Farrell, R. E.; Carverhill, J.; Lemke, R.; Knight, J. D.

    2014-12-01

    Estimates of N2O emissions in Canada indicate that 17% of all agriculture-based emissions are associated with the decomposition of crop residues. However, research specific to the western Canadian prairies (including Saskatchewan) has shown that the N2O emission factor for N sources in this region typically ranges between 0.2 and 0.6%, which is well below the current IPCC default emission factor of 1.0%. Thus, it stands to reason that emissions from crop residues should also be lower than those calculated using the current IPCC emission factor. Current data indicates that residue decomposition, N mineralization and N2O production are affected by a number of factors such as C:N ratio and chemical composition of the residue, soil type, and soil water content; thus, a bench-scale incubation study was conducted to examine the effects of soil type and water content on N2O emissions associated with the decomposition of different crop residues. The study was carried out using soils from the Black, Dark Brown, Brown, and Gray soil zones and was conducted at both 50% and 70% water-filled pore space (WFPS); the soils were amended with 15N-labeled residues of wheat, pea, canola, and flax, or with an equivalent amount of 15N-labeled urea; 15N2O production was monitored using a Picarro G5101-i isotopic N2O analyzer. Crop residue additions to the soils resulted in both direct and indirect emissions of N2O, with residue derived emissions (RDE; measured as 15N2O) generally exceeding residue-induced emissions (RIE) at 50% WFPS—with RDEs ranging from 42% to 88% (mean = 58%) of the total N2O. Conversely, at 70% WFPS, RDEs were generally lower than RIEs—ranging from 21% to 83% (mean = 48%). Whereas both water content and soil type had an impact on N2O production, there was a clear and consistent trend in the emission factors for the residues; i.e., emissions were always greatest for the canola residue and lowest for the wheat residue and urea fertilizer; and intermediate for pea

  9. The effects of isotope-labeled analogs on the LC-IDMS measurement by comparison of ESI responses and matrix effect of melamine, (13)C3-melamine, (13)C3+(15)N3-melamine, and (15)N3-melamine.

    PubMed

    Li, Xiu Qin; Zhang, Qing He; Yang, Zong; Li, Hong Mei; Huang, Dong Feng

    2017-05-01

    In this paper, the effect of isotope-labeled analogs on the liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) measurement was evaluated based on the comparison research of electrospray ionization responses (ESI) and matrix effect of melamine, (13)C3-melamine, (13)C3+(15)N3-melamine, and (15)N3-melamine. The isotope-labeled melamines had similar ionization efficiency with melamine in the electrospray ionization source, but the intensity of corresponding quantitative fragment ions had distinctive differences. Based on the density functional theory at the B3LYP/6-311+G** level, this phenomenon was explained very well. The rare cleavage pathways of melamine, which just could be exactly identified by (15)N-labeled melamines, resulted in the difference of quantitative fragment ions between (15)N-labeled melamines and melamine. The interaction of ESI response between melamine and isotope-labeled melamines was investigated using MRM monitor mode. (15)N-labeled melamine had significant ion inter-suppression effect on melamine, while (13)C-labeled melamine had little influence on melamine. Finally, the influence of different isotope-labeled melamines on the LC-IDMS result was evaluated using the IDMS correction factor (θ). Taking the determination of melamine in milk powder as an example, the matrix effects of different isotope-labeled melamines and melamine had notable difference and the impact of this difference on the measurement results depended on the concentrations of analyte and matrix solution. It was worth noting that (15)N3-melamine exhibited significant ion suppression to melamine in matrix solution. The deviation of the results from IDMS method might reach 59% using (15)N3-melamine as internal standard in special matrix solution. Graphical Abstract The comparison of ESI responses of melamine, (13)C3-melamine, (13)C3+(15)N3-melamine and (15)N3-melamine.

  10. Novel labeling technique illustrates transfer of 15N2 from Sphagnum moss to vascular plants via diazotrophic nitrogen fixation

    NASA Astrophysics Data System (ADS)

    Thorp, N. R.; Vile, M. A.; Wieder, R.

    2013-12-01

    We used 15N2 gas to trace nitrogen (N) from biological N2-fixation to vascular plant uptake in an Alberta bog in order to determine if neighboring bog plants acquire recently fixed N from diazotrophs associating with Sphagnum mosses. Recent evidence indicates high rates of N2-fixation in Sphagnum mosses of Alberta bogs (Vile et al. 2013). Our previous work has shown that mosses can assimilate fixed N from associated diazotrophs as evidenced by the high N content of mosses despite minimal inputs from atmospheric deposition, retranslocation, and N mineralization. Therefore, the potential exists for vascular plants to obtain N from ';leaky' tissues of live mosses, however, this phenomenon has not been tested previously. Here we document the potential for relatively rapid transfer to vascular plants of N fixed by Sphagnum moss-associated diazotrophs. We utilized the novel approach of incubating mosses in 15N2 to allow the process of diazotrophic N2-fixation to mechanistically provide the 15N label, which is subsequently transferred to Sphagnum mosses. The potential for vascular bog natives to tap this N was assessed by planting the vascular plants in the labeled moss. Sphagnum mosses (upper 3 cm of live plants) were incubated in the presence of 98 atom % 15N2 gas for 48 hours. Two vascular plants common to Alberta bogs; Picea mariana and Vaccinium oxycoccus were then placed in the labeled mosses, where the mosses served as the substrate. Tissue samples from these plants were collected at three time points during the incubation; prior to 15N2 exposure (to determine natural abundance 15N), and at one and two months after 15N2 exposure. Roots and leaves were separated and run separately on a mass spectrometer to determine 15N concentrations. Sphagnum moss capitula obtained N from N2-fixation (δ15N of -2.43 × 0.40, 122.76 × 23.78, 224.92 × 68.37, 143.74 × 54.38 prior to, immediately after, and at 1 and 2 months after exposure to 15N2, respectively). Nitrogen was

  11. Applicability of a "Multi-Stage Pulse Labeling" (15)N Approach to Phenotype N Dynamics in Maize Plant Components during the Growing Season.

    PubMed

    de Oliveira Silva, Amanda; Camberato, James J; Coram, Tristan; Filley, Timothy; Vyn, Tony J

    2017-01-01

    Highlights This work utilizes "multi-stage pulse labeling" (15)N applications, primarily during reproductive growth stages, as a phenotyping strategy to identify maize hybrids with superior N use efficiency (NUE) under low N conditions. Research using labeled isotopic N ((15)N) can precisely quantify fertilizer nitrogen (N) uptake and organ-specific N allocation in field crops such as maize (Zea mays L.). The overall research objective was to study plant N uptake patterns potentially correlated with N use efficiency (NUE) in field-grown maize hybrids using a "multi-stage pulse labeling" (15)N phenotyping strategy with an emphasis on the reproductive period. Five hybrids varying in NUE were compared under zero N fertilizer application (0N) plus a moderate rate of 112 kg N ha(-1) (112N) in 2013 (2 locations) and 2014 growing seasons. The equivalent of 3.2 (2013) to 2.1 (2014) kg of (15)N ha(-1), as labeled Ca((15)NO3)2, was injected into soil on both sides of consecutive plants at multiple stages between V14 and R5. Aboveground plant biomass was primarily collected in short-term intervals (4-6 days after each (15)N application) in both years, and following a single long-term interval (at R6 after (15)N injection at R1) in 2014. Averaged across hybrids and site-years, the moderate N rate (112N) increased absolute (15)N uptake at all stages; however, plants in the 0N treatment allocated proportionally more (15)N to reproductive organs. Before flowering, short-term recovery of (15)N ((15)Nrec) totaled ~0.30 or 0.40 kg kg(-1) of the (15)N applied, and ~50% of that accumulated (15)Nu was found in leaves and 40% in stems. After flowering, plant (15)Nrec totaled ~0.30 kg kg(-1) of (15)N applied, and an average 30% of accumulated (15)Nu was present in leaves, 17% in stems, and the remainder-usually the majority-in ears. At the R5 stage, despite a declining overall rate of (15)N uptake per GDD thermal unit, plant (15)Nrec represented ~0.25 kg kg(-1) of (15)N applied, of which

  12. Cost-effective production of 13C, 15N stable isotope-labelled biomass from phototrophic microalgae for various biotechnological applications.

    PubMed

    Acién Fernández, F G; Fernández Sevilla, J M; Egorova-Zachernyuk, T A; Molina Grima, E

    2005-12-01

    The present study outlines a process for the cost-effective production of 13C/15N-labelled biomass of microalgae on a commercial scale. The core of the process is a bubble column photobioreactor with exhaust gas recirculation by means of a low-pressure compressor. To avoid accumulation of dissolved oxygen in the culture, the exhaust gas is bubbled through a sodium sulphite solution prior to its return to the reactor. The engineered system can be used for the production of 13C, 15N, and 13C-15N stable isotope-labelled biomass as required. To produce 13C-labelled biomass, 13CO2 is injected on demand for pH control and carbon supply, whereas for 15N-labelled biomass Na15NO3 is supplied as nitrogen source at the stochiometric concentration. The reactor is operated in semicontinuous mode at different biomass concentrations, yielding a maximum mean biomass productivity of 0.3 gL(-1) day(-1). In order to maximize the uptake efficiency of the labelled substrates, the inorganic carbon is recovered from the supernatant by acidification/desorption processes, while the nitrate is delivered at stochiometric concentration and the harvesting of biomass is performed when the 15NO3- is depleted. In these conditions, elemental analysis of both biomass and supernatant shows that 89.2% of the injected carbon is assimilated into the biomass and 6.9% remains in the supernatant. Based on elemental analysis, 97.8% of the supplied nitrogen is assimilated into the biomass and 1.3% remains in the supernatant. Stable isotope-labelling enrichment has been analysed by GC-MS results showing that the biomass is highly labelled. All the fatty acids are labelled; more than 96% of the carbon present in these fatty acids is 13C. The engineered system was stably operated for 3 months, producing over 160 g of 13C and/or 15N-labelled biomass. The engineered bioreactor can be applied for the labelling of various microalgae.

  13. High retention of (15) N-labeled nitrogen deposition in a nitrogen saturated old-growth tropical forest.

    PubMed

    Gurmesa, Geshere Abdisa; Lu, Xiankai; Gundersen, Per; Mao, Qinggong; Zhou, Kaijun; Fang, Yunting; Mo, Jiangming

    2016-11-01

    The effects of increased reactive nitrogen (N) deposition in forests depend largely on its fate in the ecosystems. However, our knowledge on the fates of deposited N in tropical forest ecosystems and its retention mechanisms is limited. Here, we report the results from the first whole ecosystem (15) N labeling experiment performed in a N-rich old-growth tropical forest in southern China. We added (15) N tracer monthly as (15) NH4(15) NO3 for 1 year to control plots and to N-fertilized plots (N-plots, receiving additions of 50 kg N ha(-1)  yr(-1) for 10 years). Tracer recoveries in major ecosystem compartments were quantified 4 months after the last addition. Tracer recoveries in soil solution were monitored monthly to quantify leaching losses. Total tracer recovery in plant and soil (N retention) in the control plots was 72% and similar to those observed in temperate forests. The retention decreased to 52% in the N-plots. Soil was the dominant sink, retaining 37% and 28% of the labeled N input in the control and N-plots, respectively. Leaching below 20 cm was 50 kg N ha(-1)  yr(-1) in the control plots and was close to the N input (51 kg N ha(-1)  yr(-1) ), indicating N saturation of the top soil. Nitrogen addition increased N leaching to 73 kg N ha(-1)  yr(-1) . However, of these only 7 and 23 kg N ha(-1)  yr(-1) in the control and N-plots, respectively, originated from the labeled N input. Our findings indicate that deposited N, like in temperate forests, is largely incorporated into plant and soil pools in the short term, although the forest is N-saturated, but high cycling rates may later release the N for leaching and/or gaseous loss. Thus, N cycling rates rather than short-term N retention represent the main difference between temperate forests and the studied tropical forest.

  14. Determination of the mutual orientation of the 15N and 13C NMR chemical shift tensors of 13- 15N double labeled model peptides for silk fibroin from the dipolar-coupled powder patterns

    NASA Astrophysics Data System (ADS)

    Asakura, Tetsuo; Yamazaki, Yasunobu; Seng, Koo Wey; Demura, Makoto

    1998-05-01

    The 15N and 13C chemical shift tensors, and the orientation of the principal axis system relative to the molecular symmetry axes were determined for 15N and 13C carbonyl carbon sites of 13C 15N double labeled model peptides for Bombyx mori silk fibroin, that is, Boc-[1- 13C]Ala[ 15N]Gly-OMe, Boc-[1- 13C]Ala[ 15N]GlyAlaGly-OPac, Boc-AlaGly[1- 13C]Ala[ 15N]GlyAlaGly-OPac, Boc-[1- 13C]Gly[ 15N]AlaGlyAla-OPac, Boc-GlyAla[1- 13C]Gly[ 15N]AlaGlyAla-OPac and Boc-[1- 13C]Gly[ 15N]ValGlyAla-OPac, where Boc is t-butoxycarbonyl, OMe is methyl ester, OPac is phenacyl ester, Ala is alanine, Gly is glycine and Val is valine. From the comparisons of the 15N chemical shift tensors and the orientations of the principal axis system relative to the molecular symmetry axes among three compounds having [1- 13C]Ala[ 15N]Gly units, it is concluded that the intermolecular interactions such as hydrogen bonding are different between Boc-[1- 13C]Ala[ 15N]Gly-OMe and two compounds, Boc-[1- 13C]Ala[ 15N]GlyAlaGly-OPac and Boc-AlaGly[1- 13C]Ala[ 15N]GlyAlaGly-OPac although the latter two compounds have similar structures. A similar conclusion has also been obtained from the 13C chemical shift tensors of these compounds.

  15. High Resolution 13C MRI With Hyperpolarized Urea: In Vivo T2 Mapping and 15N Labeling Effects

    PubMed Central

    Reed, Galen D.; von Morze, Cornelius; Bok, Robert; Koelsch, Bertram L.; Van Criekinge, Mark; Smith, Kenneth J.; Shang, Hong; Larson, Peder E. Z.; Kurhanewicz, John; Vigneron, Daniel B.

    2014-01-01

    13C steady state free precession (SSFP) magnetic resonance imaging and effective spin-spin relaxation time (T2) mapping were performed using hyperpolarized [13C] urea and [13C, 15N2] urea injected intravenously in rats. 15N labeling gave large T2 increases both in solution and in vivo due to the elimination of a strong scalar relaxation pathway. The T2 increase was pronounced in the kidney, with [13C, 15N2] urea giving T2 values of 6.3±1.3 s in the cortex and medulla, and 11±2 s in the renal pelvis. The measured T2 in the aorta was 1.3±0.3 s. [13C] urea showed shortened T2 values in the kidney of 0.23±0.03 s compared to 0.28±0.03 s measured in the aorta. The enhanced T2 of [13C, 15N2] urea was utilized to generate large signal enhancement by SSFP acquisitions with flip angles approaching the fully refocused regime. Projection images at 0.94 mm in-plane resolution were acquired with both urea isotopes, with [13C, 15N2] urea giving a greater than four-fold increase in signal-to-noise ratio [13C] over urea. PMID:24235273

  16. Fungal functioning in a pine forest: evidence from a 15N-labeled global change experiment

    Treesearch

    Erik A. Hobbie; Linda T.A. van Diepen; Erik A. Lilleskov; Andrew P. Oiumette; Adrien C. Finzi; Kirsten S. Hofmockel

    2014-01-01

    We used natural and tracer nitrogen (N) isotopes in a Pinus taeda free air CO2 enrichment (FACE) experiment to investigate functioning of ectomycorrhizal and saprotrophic fungi in N cycling. Fungal sporocarps were sampled in 2004 (natural abundance and 15N tracer) and 2010 (tracer) and δ15...

  17. Coupling Sap Flow Velocity and Amino Acid Concentrations as an Alternative Method to 15N Labeling for Quantifying Nitrogen Remobilization by Walnut Trees1

    PubMed Central

    Frak, Ela; Millard, Peter; Le Roux, Xavier; Guillaumie, Sabine; Wendler, Renate

    2002-01-01

    The temporal dynamics of N remobilization was studied in walnut (Juglans nigra × regia) trees growing in sand culture. Trees were fed with labeled N (15N) during 1999 and unlabeled N in 2000. Total N and 15N contents in different tree compartments were measured during 80 d after bud burst and were used to estimate N remobilization for spring growth. The seasonal (and occasionally diurnal) dynamics of the concentration and 15N enrichment of the major amino acids in xylem sap were determined concurrently. Sap flow velocity was also measured for sample trees. A new approach coupling amino acid concentrations to sap flow velocity for quantifying N remobilization was tested. A decrease of the labeled N contents of medium roots, tap roots, and trunk was observed concurrently to the increase in the labeled N content of new shoots. Remobilized N represented from previous year storage 54% of N recovered in new shoots. Arginine, citruline, γ-amino butyric acid, glutamic acid, and aspartic acid always represented around 80% of total amino acid and amide N in xylem sap and exhibited specific seasonal trends and significant diurnal trends. N translocation was mainly insured by arginine during the first 15 d after bud burst, and then by glutamic acid and citruline. The pattern of N remobilization estimated by the new approach was consistent with that measured by the classical labeling technique. Implications for quantifying N remobilization for large, field-growing trees are discussed. PMID:12376667

  18. Coupling sap flow velocity and amino acid concentrations as an alternative method to (15)N labeling for quantifying nitrogen remobilization by walnut trees.

    PubMed

    Frak, Ela; Millard, Peter; Le Roux, Xavier; Guillaumie, Sabine; Wendler, Renate

    2002-10-01

    The temporal dynamics of N remobilization was studied in walnut (Juglans nigra x regia) trees growing in sand culture. Trees were fed with labeled N ((15)N) during 1999 and unlabeled N in 2000. Total N and (15)N contents in different tree compartments were measured during 80 d after bud burst and were used to estimate N remobilization for spring growth. The seasonal (and occasionally diurnal) dynamics of the concentration and (15)N enrichment of the major amino acids in xylem sap were determined concurrently. Sap flow velocity was also measured for sample trees. A new approach coupling amino acid concentrations to sap flow velocity for quantifying N remobilization was tested. A decrease of the labeled N contents of medium roots, tap roots, and trunk was observed concurrently to the increase in the labeled N content of new shoots. Remobilized N represented from previous year storage 54% of N recovered in new shoots. Arginine, citruline, gamma-amino butyric acid, glutamic acid, and aspartic acid always represented around 80% of total amino acid and amide N in xylem sap and exhibited specific seasonal trends and significant diurnal trends. N translocation was mainly insured by arginine during the first 15 d after bud burst, and then by glutamic acid and citruline. The pattern of N remobilization estimated by the new approach was consistent with that measured by the classical labeling technique. Implications for quantifying N remobilization for large, field-growing trees are discussed.

  19. Quantitative analysis of 15N labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives

    USDA-ARS?s Scientific Manuscript database

    The enteral metabolism of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of 15N labeled glutamine results in the incorporation of the 15N label into cit...

  20. Importance of bacterivory and preferential selection toward diatoms in larvae of Crepidula fornicata (L.) assessed by a dual stable isotope (13C, 15N) labeling approach

    NASA Astrophysics Data System (ADS)

    Leroy, Fanny; Riera, Pascal; Jeanthon, Christian; Edmond, Frédérique; Leroux, Cédric; Comtet, Thierry

    2012-05-01

    In Europe, the gastropod Crepidula fornicata is an invasive species characterized by a long reproductive period (from February to November). Thus, its larvae are exposed to variations in available food sources (in terms of quantity and quality). We aimed to investigate if bacteria could contribute to larval food both in presence or absence of phytoplankton, and to compare these results to seasonal variations of bacteria and phytoplankton abundances at a coastal site in the English Channel. First, ingestion of fluorescent beads of 0.5 to 2 μm diameter, showed that larvae were able to ingest particles of typical bacterial size. Then we used a dual stable isotope labeling approach which consisted in labeling a bacterial pelagic community with 15N and a diatom (Chaetoceros gracilis) culture with 13C, and supplying larvae with 15N-labeled bacteria, 13C-labeled diatoms, and both labeled sources. This technique has, to our knowledge, never been applied to invertebrate larvae. After 24 h of experiment, larvae were significantly enriched in all treatments: + 21.5‰ (∆δ13C) when supplied with diatoms, + 1364‰ (∆δ15N) when supplied with bacteria, and + 24‰ (∆δ13C) and + 135‰ (∆δ15N) when supplied with the two mixed sources. These results indicated that bacteria can contribute to the larval nutrition in C. fornicata, even in the presence of phytoplankton. Our results however suggested that larvae of C. fornicata preferentially used diatoms and showed that the supply of free bacteria did not alter the uptake of diatoms. Considering the seasonal variations of bacteria and phytoplankton abundances at the study site, these results suggested that bacteria may constitute a complementary resource for the larvae of C. fornicata when phytoplankton is abundant and may become a substitute resource when phytoplankton is less available. This approach offers promising perspectives to trace food sources and assess nitrogen and carbon fluxes between planktotrophic larvae

  1. The Contamination of Commercial 15N2 Gas Stocks with 15N–Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements

    PubMed Central

    Dabundo, Richard; Lehmann, Moritz F.; Treibergs, Lija; Tobias, Craig R.; Altabet, Mark A.; Moisander, Pia H.; Granger, Julie

    2014-01-01

    We report on the contamination of commercial 15-nitrogen (15N) N2 gas stocks with 15N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. 15N2 gas is used to estimate N2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled 15N2 into organic matter. However, the microbial assimilation of bioavailable 15N-labeled N2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N2 fixation rates. 15N2 gas procured from three major suppliers was analyzed for the presence of these 15N-contaminants. Substantial concentrations of 15N-contaminants were detected in four Sigma-Aldrich 15N2 lecture bottles from two discrete batch syntheses. Per mole of 15N2 gas, 34 to 1900 µmoles of 15N-ammonium, 1.8 to 420 µmoles of 15N-nitrate/nitrite, and ≥21 µmoles of 15N-nitrous oxide were detected. One 15N2 lecture bottle from Campro Scientific contained ≥11 µmoles of 15N-nitrous oxide per mole of 15N2 gas, and no detected 15N-nitrate/nitrite at the given experimental 15N2 tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles 15N-nitrous oxide per mole 15N2, and trace concentrations of 15N-ammonium and 15N-nitrate/nitrite. 15N2 gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the 15N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N2 fixation assays suggests that the degree of detected 15N-ammonium contamination could yield inferred N2 fixation rates ranging from undetectable, <0.01 nmoles N L−1 d−1, to 530 nmoles N L−1 d−1, contingent on experimental conditions. These rates are comparable to, or greater than, N2 fixation rates commonly detected in field assays. These results indicate that past reports of N2 fixation should be interpreted with caution, and demonstrate that the purity of commercial 15N2

  2. An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids.

    PubMed

    Nikonowicz, E P; Pardi, A

    1993-08-20

    An efficient method is presented for the assignment of the proton, carbon, and nitrogen resonances in the NMR spectra of isotopically labeled nucleic acids. The assignment strategy starts by identifying all protons and carbons belonging to the same sugar ring through application of a set of 2D or 3D heteronuclear HCCH NMR experiments. Next the individual sugar rings are connected to their corresponding bases through intra-residue 1H-1H nuclear Overhauser effects (NOEs) observed in a 3D (1H, 13C, 1H) NOESY-HMQC experiment. Sequential NOE connectivities observed in this experiment are then used to assign each residue in the nucleotide sequence. The imino protons and nitrogens, and the cytidine amino protons and nitrogens, are assigned by 2D (15N, 1H) HMQC and 3D (1H, 15N, 1H) NOESY-HMQC experiments in H2O. This assignment procedure is illustrated on the 99% 13C/15N labeled RNA duplex r(GGCGCUUGCGUC)2. The application of these multi-dimensional heteronuclear magnetic resonance experiments enormously simplifies the resonance assignment of nucleic acids and allows assignment of many more protons, carbons and nitrogens than was possible using standard techniques on unlabeled molecules. Since a larger percentage of the protons can now be assigned by these experiments, much more NMR structural information can be obtained which will significantly extend the size limit for solution structure determinations of RNAs.

  3. Decomposition and nitrogen dynamics of (15)N-labeled leaf, root, and twig litter in temperate coniferous forests.

    PubMed

    van Huysen, Tiff L; Harmon, Mark E; Perakis, Steven S; Chen, Hua

    2013-12-01

    Litter nutrient dynamics contribute significantly to biogeochemical cycling in forest ecosystems. We examined how site environment and initial substrate quality influence decomposition and nitrogen (N) dynamics of multiple litter types. A 2.5-year decomposition study was installed in the Oregon Coast Range and West Cascades using (15)N-labeled litter from Acer macrophyllum, Picea sitchensis, and Pseudotsuga menziesii. Mass loss for leaf litter was similar between the two sites, while root and twig litter exhibited greater mass loss in the Coast Range. Mass loss was greatest from leaves and roots, and species differences in mass loss were more prominent in the Coast Range. All litter types and species mineralized N early in the decomposition process; only A. macrophyllum leaves exhibited a net N immobilization phase. There were no site differences with respect to litter N dynamics despite differences in site N availability, and litter N mineralization patterns were species-specific. For multiple litter × species combinations, the difference between gross and net N mineralization was significant, and gross mineralization was 7-20 % greater than net mineralization. The mineralization results suggest that initial litter chemistry may be an important driver of litter N dynamics. Our study demonstrates that greater amounts of N are cycling through these systems than may be quantified by only measuring net mineralization and challenges current leaf-based biogeochemical theory regarding patterns of N immobilization and mineralization.

  4. Decomposition and nitrogen dynamics of 15N-labeled leaf, root, and twig litter in temperate coniferous forests

    USGS Publications Warehouse

    van Huysen, Tiff L.; Harmon, Mark E.; Perakis, Steven S.; Chen, Hua

    2013-01-01

    Litter nutrient dynamics contribute significantly to biogeochemical cycling in forest ecosystems. We examined how site environment and initial substrate quality influence decomposition and nitrogen (N) dynamics of multiple litter types. A 2.5-year decomposition study was installed in the Oregon Coast Range and West Cascades using 15N-labeled litter from Acer macrophyllum, Picea sitchensis, and Pseudotsuga menziesii. Mass loss for leaf litter was similar between the two sites, while root and twig litter exhibited greater mass loss in the Coast Range. Mass loss was greatest from leaves and roots, and species differences in mass loss were more prominent in the Coast Range. All litter types and species mineralized N early in the decomposition process; only A. macrophyllum leaves exhibited a net N immobilization phase. There were no site differences with respect to litter N dynamics despite differences in site N availability, and litter N mineralization patterns were species-specific. For multiple litter × species combinations, the difference between gross and net N mineralization was significant, and gross mineralization was 7–20 % greater than net mineralization. The mineralization results suggest that initial litter chemistry may be an important driver of litter N dynamics. Our study demonstrates that greater amounts of N are cycling through these systems than may be quantified by only measuring net mineralization and challenges current leaf-based biogeochemical theory regarding patterns of N immobilization and mineralization.

  5. Heavy water and 15N labeling with NanoSIMS analysis reveals growth-rate dependent metabolic heterogeneity in chemostats

    PubMed Central

    McGlynn, Shawn E.; Green-Saxena, Abigail

    2015-01-01

    To measure single cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labeling of microbial populations with heavy water (a passive tracer) and 15N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % 2H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 hours, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium vs. amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments. PMID:25655651

  6. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C, 15N-labeled peptides and proteins

    NASA Astrophysics Data System (ADS)

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly 13C, 15N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22 kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600 MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i - 2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.

  7. A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

    PubMed Central

    Trompelt, Kerstin; Steinbeck, Janina; Terashima, Mia; Hippler, Michael

    2014-01-01

    The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions. PMID:24686495

  8. Application of rate equations to ELDOR and saturation recovery experiments on 14N: 15N spin-label pairs

    NASA Astrophysics Data System (ADS)

    Yin, Jun-Jie; Hyde, James S.

    Rate equations describing the time dependence of population differences of the five allowed transitions in an 14N 15N spin-label pair problem are set up. Included in the formulation are the three Heisenberg exchange rate constants and different nitrogen nuclear spin-lattice relaxation rates, electron spin-lattice relaxation rates, and populations for the 14N and 15N moieties. Using matrix algebra, stationary and time-dependent solutions are obtained in a unified theoretical framework. The calculations apply to stationary and pulse electron-electron double resonance and to saturation-recovery ESR. Particular emphasis is placed on short pulse initial excitation, where the transverse relaxation processes are sufficiently slow that only the population difference of the irradiated transition departs significantly from Boltzmann equilibrium during the excitation.

  9. Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.

    PubMed

    Meola, Annalisa; Deville, Célia; Jeffers, Scott A; Guardado-Calvo, Pablo; Vasiliauskaite, Ieva; Sizun, Christina; Girard-Blanc, Christine; Malosse, Christian; van Heijenoort, Carine; Chamot-Rooke, Julia; Krey, Thomas; Guittet, Eric; Pêtres, Stéphane; Rey, Félix A; Bontems, François

    2014-10-01

    Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure.

  10. 1H and 15N Dynamic Nuclear Polarization Studies of Carbazole

    SciTech Connect

    Hu, Jian Zhi; Solum, Mark S.; Wind, Robert A.; Nilsson, Brad L.; Peterson, Matt A.; Pugmire, Ronald J.; Grant, David M.

    2000-01-01

    15N NMR experiments, combined with dynamic nuclear polarization (DNP), are reported on carbazole doped with the stable free radical 1,3 bisdiphenylene-2 phenylally1 (BDPA). Doping shortens the nuclear relaxation times and provides paramagnetic centers that can be used to enhance the nuclear signal by means of DNP so that 15 N NMR experiments can be done in minutes. The factors were measured in a 1.4 T external field, using both unlabeled and 98% 15N labeled carbazole with doping levels varying between 0.65 and 5.0 wt % BDPA. A doping level of approximately 1 wt % produced optimal results. DNP enhancement factors of 35 and 930 were obtained for 1H and 15N, respectively making it possible to perform 15N DNP NMR experiments at the natural abundance level.

  11. {sup 1}H and {sup 15}N dynamic nuclear polarization studies of carbazole

    SciTech Connect

    Hu, J.Z.; Solum, M.S.; Wind, R.A.; Nilsson, B.L.; Peterson, M.A.; Pugmire, R.J.; Grant, D.M.

    2000-05-18

    {sup 15}N NMR experiments, combined with dynamic nuclear polarization (DNP), are reported on carbazole doped with the stable free radical 1,3-bisdiphenylene-2-phenylallyl (BDPA). Doping shortens the nuclear relaxation times and provides paramagnetic centers that can be used to enhance the nuclear signal by means of DNP so that {sup 15}N NMR experiments can be done in minutes. The factors were measured in a 1.4 T external field, using both unlabeled and 98% {sup 15}N labeled carbazole with doping levels varying between 0.65 and 5.0 wt {degree} BDPA. A doping level of approximately 1 wt {degree} produced optimal results. DNP enhancement factors of 35 and 930 were obtained for {sup 1}H and {sup 15}N, respectively, making it possible to perform {sup 15}N DNP NMR experiments at the natural abundance level.

  12. Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously.

    PubMed

    Blank, Lars M; Desphande, Rahul R; Schmid, Andreas; Hayen, Heiko

    2012-06-01

    Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.

  13. Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein

    SciTech Connect

    Strzelecka, T.; Gronenborn, A.M.; Clore, G.M.

    1994-12-01

    The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.

  14. Amino acids as a nitrogen source in temperate upland grasslands: the use of dual labelled ((13)C, (15)N) glycine to test for direct uptake by dominant grasses.

    PubMed

    Streeter, T C; Bol, R; Bardgett, R D

    2000-01-01

    It is becoming increasingly apparent that soil amino acids are a principal source of nitrogen (N) for certain plants, and especially those of N-limited environments. This study of temperate upland grasslands used glycine-2-(13)C-(15)N and ((15)NH4)(2)SO(4) labelling techniques to test the hypothesis that plant species which dominate 'unimproved' semi-natural grasslands (Festuca-Agrostis-Galium) are able to utilise amino acid N for growth, whereas those plants which dominate 'improved' grasslands (Lolium-Cynosurus), that receive regular applications of inorganic fertiliser, use inorganic N forms as their main N source. Data from field experiments confirmed that 'free' amino acids were more abundant in 'unimproved' than 'improved' grassland and that glycine was the dominant amino acid type (up to 42% of total). Secondly, the injection of representative amounts of glycine-2-(13)C-(15)N (4.76 and 42.86 mM) into intact soil cores from the two grassland types provided evidence of direct uptake of glycine by plants, with both (15)N and (13)C being detected in plant material of both grasslands. Finally, a microcosm experiment demonstrated no preferential uptake of amino acid N by the grasses which dominate the grassland types, namely Holcus lanatus, Festuca rubra, Agrostis capillaris from the 'unimproved' grassland, and Lolium perenne from the 'improved' grassland. Again, both (13)C and (15)N were detected in all grass species suggesting uptake of intact glycine by these plants.

  15. The Kinetics of Intramolecular Distribution of 15N in Uric Acid after Administration of [15N]Glycine A REAPPRAISAL OF THE SIGNIFICANCE OF PREFERENTIAL LABELING OF N-(3 + 9) OF URIC ACID IN PRIMARY GOUT

    PubMed Central

    Sperling, Oded; Wyngaarden, James B.; Starmer, C. Frank

    1973-01-01

    The concept of an abnormality of glutamine metabolism in primary gout was first proposed on the basis of isotope data: when [15N]glycine was administered to gouty subjects, there was disproportionately great enrichment of N-(3 + 9) of uric acid, which derive from the amide-N of glutamine. An unduly high concentration of 15N in glutamine was postulated, and attributed to a hypothetical defect in catabolism of glutamine. Excess glutamine was proposed as the driving force of uric acid overproduction. We have reexamined this proposition in four gouty subjects: one mild overproducer of uric acid with “idiopathic gout,” one marked overproducer with high-grade but “partial” hypoxanthine-guanine phosphoribosyl-transferase deficiency, and two extraordinary overproducers with superactive phosphoribosylpyrophosphate synthetases. In the last three, the driving force of excessive purine biosynthesis is a known surplus of α-5-phosphoribosyl-1-pyrophosphate. Disproportionately high labeling of N-(3 + 9) was present in all four gouty subjects, most marked in the most flamboyant overproducers. The precursor glucine pool was sampled by periodic administration of benzoic acid and isolation of urinary hippuric acid. Similarly, the precursor glutamine pool was sampled by periodic administration of phenylacetic acid and isolation of the amide-N of urinary phenylacetylglutamine. The time course of 15N enrichment of hippurate differed from that of the amide-N of glutamine. Whereas initial enrichment values of hippurate were very high, those of glutamine-amide-N were low, increasing to a maximum at about 3 h, and then declining less rapidly than those of hippurate. However, enrichment values of hippurate and of phenacetyl glutamine were normal in all of the gouty subjects studied. Thus, preferential enrichment of N-(3 + 9) in gouty overproducers given [15N]glycine does not necessarily reflect a specific abnormality of glutamine metabolism, but rather appears to be a kinetic

  16. Nitrous oxide emissions from soil amended with 15N-labelled urea with nitrification inhibitor (Nitrapyrin) and mulch

    NASA Astrophysics Data System (ADS)

    Khan, Aamir; Heiling, Maria; Zaman, Mohammad; Resch, Christian

    2017-04-01

    Nitrous oxide (N2O), one of the key greenhouse and ozone (O3) depleting gases, constitutes 7% of the anthropogenic greenhouse effect. Its global warming potential is 310 times higher than that of carbon dioxide (CO2) and 16 times than methane (CH4) over a 100-year period. To develop mitigation tools for N2O emissions, and to investigate the relationship between gross N transformation and N2O emission from soil, it is imperative to understand N2O emission from soils as influenced by N inputs, environmental conditions and farm management practices. The use of nitrification inhibitor such as Nitrapyrin and crop residues (mulch) may have a role in mitigating N2O losses from soil because of their effects on nitrification and denitrification. It prevents hydrolytic action on urea and keeps nitrogen in ammonium form. To determine the effects of urea applied with nitrification inhibitor and mulch on N2O emissions from soil, an incubation experiment was conducted under controlled moisture of 60% water filled pore space (WFPS) and temperature (20±2oC) conditions. Soil samples (0-20 cm soil depth) collected from an arable site were treated with 15N-labelled urea (5 atom %) at 150 kg N/ha rate. The 5 treatments including control, (urea, urea with Nitrapyrin (800 g/100 kg urea), urea with mulch (5 tons/ha) and urea with Nitrapyrin and mulch) were replicated 4 times using 500 ml glass jars. The N2O isotopic signature and the intramolecular distribution of 15N were measured by off-axis integrated cavity output spectroscopy (Los Gatos Research). The preliminary results showed that nitrification inhibitor (Nitrapyrin) can be used to distinguish between different pathways of N2O production from soil. In addition to the site preference of the 15N promises to be a helpful tool to determine the source of the generated N2O.

  17. Covalent binding of aniline to humic substances. 2. 15N NMR studies of nucleophilic addition reactions

    USGS Publications Warehouse

    Thorn, K.A.; Pettigrew, P.J.; Goldenberg, W.S.; Weber, E.J.

    1996-01-01

    Aromatic amines are known to undergo covalent binding with humic substances in the environment. Although previous studies have examined reaction conditions and proposed mechanisms, there has been no direct spectroscopic evidence for the covalent binding of the amines to the functional groups in humic substances. In order to further elucidate the reaction mechanisms, the Suwannee River and IHSS soil fulvic and humic acids were reacted with 15N-labeled aniline at pH 6 and analyzed using 15N NMR spectrometry. Aniline underwent nucleophilic addition reactions with the quinone and other carbonyl groups in the samples and became incorporated in the form of anilinohydroquinone, anilinoquinone, anilide, imine, and heterocyclic nitrogen, the latter comprising 50% or more of the bound amine. The anilide and anilinohydroquinone nitrogens were determined to be susceptible to chemical exchange by ammonia. In the case of Suwannee River fulvic acid, reaction under anoxic conditions and pretreatment with sodium borohydride or hydroxylamine prior to reaction under oxic conditions resulted in a decrease in the proportion of anilinohydroquinone nitrogen incorporated. The relative decrease in the incorporation of anilinohydroquinone nitrogen with respect to anilinoquinone nitrogen under anoxic conditions suggested that inter- or intramolecular redox reactions accompanied the nucleophilic addition reactions.

  18. Potential probe for examining opiate-receptor interactions: model compound study of dynamic effects on /sup 15/N INEPT enhancements

    SciTech Connect

    Schilling, K.H.; Mikita, M.A.

    1987-10-01

    Model systems were chosen in an attempt to mimic the proton exchange environment of an agonist nitrogen in an opiate-receptor interaction. The two model systems studied were an ammonium: 18-crown-6 ether complex and a quinuclidine-trifluoroacetic acid ion pair. Each system was examined for their effects on /sup 15/N NMR INEPT enhancements. Both models were found to retard proton exchange dynamics, as observed by increased enhancements relative to free ions in neutral aqueous solutions. These results suggest that the confinement of a protonated nitrogen, such as that expected in receptor binding, may alter exchange dynamics to favor INEPT enhancements, while unbound agonists would remain unenhanced. As a result, /sup 15/N NMR INEPT enhancements from a solution of receptor subtypes with an appropriate /sup 15/N-labeled agonist may present a means of exploring the dynamics of direct opiate-receptor interactions.

  19. 15N-tracer studies in formula-fed preterm infants: the role of glycine supply in protein turnover.

    PubMed

    Plath, C; Heine, W; Wutzke, K D; Uhlemann, M

    1996-10-01

    In preterm infants, protein-turnover rates obtained by [15N]glycine as a tracer are known to be overestimated. This may reflect the insufficient supply of dietary glycine. In this randomized study, the influence of a glycine-rich diet on whole body protein turnover rates in eight male preterm infants (29-32 weeks, 1,200-2,540 g birthweight) using the 15N-tracer technique on days 21 and 28 of life was investigated to evaluate the necessity of supplementing preterm infant formulas with proteins rich in glycine. Before and during the study, the infants were alternately fed with a commercial available preterm infant formula (I, 2% protein, 40 mg glycine/dl) and a variety of this formulation with glycine-rich proteins (II, 2% protein, 130 mg glycine/dl). The protein-turnover rates were computed after 15N-single-pulse labeling with the help of the three-compartment model (TCM) and the urinary ammonia end-product method (AEPM). The tracer used was [15N]glycine (dosage: 2 mg 15N/kg). For the determination of 15N-excess-excretion kinetics, fractionated urine specimens were collected over a 36-h period. The protein-turnover rate calculated by TCM was 8.8 +/- 1.6 g/kg/day (formula I) and 7.7 +/- 2.0 g/kg/day (formula II); using AEPM, the rate was 8.7 +/- 2.5 g/kg/day and 7.5 +/- 1.5 g/kg/day, respectively. We conclude that the presaturation of the precursor pool by an adequate glycine intake minimizes drawbacks that may arise when using [15N]-glycine as a tracer in preterm infants, and a protein concentration of 2%, as in formula I, and consequently, a 170% glycine content when compared with the same human milk volume, meets the glycine requirement.

  20. [Absorption and distribution of nitrogen from 15N labelled urea applied at core-hardening stage in winter jujube].

    PubMed

    Zhao, Dengchao; Jiang, Yuanmao; Peng, Futian; Zhang, Jin; Zhang, Xu; Ju, Xiaotang; Zhang, Fusuo

    2006-01-01

    The study with pot experiment showed that at the rapid-swelling stage of winter jujube fruit, the percent of nitrogen derived from fertilizer (Ndff%) was the highest (10.64%) in fine roots, followed by new-growth nutritive organs. The absorbed urea-15N decreased in leaves and deciduous supers, and accumulated preferentially in root systems after harvest. The Ndff% in coarse roots was the highest (3.69%) before budding stage, while that in new-growth organs (new branches, deciduous supers, leaves and flowers) was the highest at full-blooming stage. The urea-15N applied at core-hardening stage mainly allocated in nutritive organs (leaves, deciduous supers, roots) in the first year, with the distribution rate 54.01% in root systems in winter, which was higher than that in branches (45.99%). The 15N stored in main branches changed drastically from post-harvest to budding stage. Main branches could be regarded as the 'target organs' of N storage, while coarse roots were the 'long-term sink' of N storage. The N reserve distributed preferentially in contiguity organs, and the distribution center changed with the growth and development of winter jujube in next spring.

  1. Multi-Isotope Secondary Ion Mass Spectrometry Combining Heavy Water 2H with 15N Labeling As Complementary Tracers for Metabolic Heterogeneity at the Single-Cell Level

    NASA Astrophysics Data System (ADS)

    Kopf, S.; McGlynn, S.; Cowley, E.; Green, A.; Newman, D. K.; Orphan, V. J.

    2014-12-01

    Metabolic rates of microbial communities constitute a key physiological parameter for understanding the in situ growth constraints for life in any environment. Isotope labeling techniques provide a powerful approach for measuring such biological activity, due to the use of isotopically enriched substrate tracers whose incorporation into biological materials can be detected with high sensitivity by isotope-ratio mass spectrometry. Nano-meter scale secondary ion mass spectrometry (NanoSIMS) combined with stable isotope labeling provides a unique tool for studying the spatiometabolic activity of microbial populations at the single cell level in order to assess both community structure and population diversity. However, assessing the distribution and range of microbial activity in complex environmental systems with slow-growing organisms, diverse carbon and nitrogen sources, or heterotrophic subpopulations poses a tremendous technical challenge because the introduction of isotopically labeled substrates frequently changes the nutrient availability and can inflate or bias measures of activity. Here, we present the use of hydrogen isotope labeling with deuterated water as an important new addition to the isotopic toolkit and apply it for the determination of single cell microbial activities by NanoSIMS imaging. This tool provides a labeling technique that minimally alters any aquatic chemical environment, can be administered with strong labels even in minimal addition (natural background is very low), is an equally universal substrate for all forms of life even in complex, carbon and nitrogen saturated systems, and can be combined with other isotopic tracers. The combination of heavy water labeling with the most commonly used NanoSIMS tracer, 15N, is technically challenging but opens up a powerful new set of multi-tracer experiments for the study of microbial activity in complex communities. We present the first truly simultaneous single cell triple isotope system

  2. Development of equations to estimate microbial contamination in ruminal incubation residues of forage produced under tropical conditions using 15N as a label.

    PubMed

    Machado, P A S; Valadares Filho, S C; Detmann, E; Santos, S A; Valadares, R F D; Ducatti, C; Rotta, P P; Costa e Silva, L F

    2013-08-01

    The objective of this study was to use (15)N to label microbial cells to allow development of equations for estimating the microbial contamination in ruminal in situ incubation residues of forage produced under tropical conditions. A total of 24 tropical forages were ruminal incubated in 3 steers at 3 separate times. To determine microbial contamination of the incubated residues, ruminal bacteria were labeled with (15)N by continuous intraruminal infusion 60 h before the first incubation and continued until the last day of incubation. Ruminal digesta was collected for the isolation of bacteria before the first infusion of (15)N on adaptation period and after the infusion of (15)N on collection period. To determine the microbial contamination of CP fractions, restricted models were compared with the full model using the model identity test. A value of the corrected fraction "A" was estimated from the corresponding noncorrected fraction by this equation: Corrected "A" fraction (A(CP)C) = 1.99286 + 0.98256 × A" fraction without correction (A(CP)WC). The corrected fraction "B" was estimated from the corresponding noncorrected fraction and from CP, NDF, neutral detergent insoluble protein (NDIP), and indigestible NDF (iNDF) using the equation corrected "B" fraction (B(CP)C) = -17.2181 - 0.0344 × fraction "B" without correction (B(CP)WC) + 0.65433 × CP + 1.03787 × NDF + 2.66010 × NDIP - 0.85979 × iNDF. The corrected degradation rate of "B" fraction (kd)was estimated using the equation corrected degradation rate of "B" fraction (kd(CP)C) = 0.04667 + 0.35139 × degradation rate of "B" fraction without correction (kd(CP)WC) + 0.0020 × CP - 0.00055839 × NDF - 0.00336 × NDIP + 0.00075089 × iNDF. This equation was obtained to estimate the contamination using CP of the feeds: %C = 79.21 × (1 - e(-0.0555t)) × e(-0.0874CP). It was concluded that A and B fractions and kd of CP could be highly biased by microbial CP contamination, and therefore these corrected values

  3. Chemical synthesis of glycoproteins with the specific installation of gradient enriched 15N-labeled amino acids for getting insight into glycoprotein behavior.

    PubMed

    Kajihara, Yasuhiro; Nguyen, Minh Hien; Izumi, Masayuki; Sato, Hajime; Okamoto, Ryo

    2017-03-09

    We propose a novel partially 15N-labelling method for the amide backbone of a synthetic glycoprotein. By use of a chemical approach utilizing SPPS and NCL, we inserted thirteen 15N-labeled amino acids at specific positions of the protein backbone, while intentionally varying the enrichment of 15N atoms. This idea enables us to discriminate even the same type of amino acid based on the intensities of 1H-15N HSQC signals, thus allowing us to understand the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied CD spectra and T1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure.

  4. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone ((1)H(N), (15)N and (13)C') resonances in (15)N/(13)C-labeled proteins.

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin. Copyright © 2011 John Wiley & Sons, Ltd.

  5. Dynamics of amino acid redistribution in the carnivorous Venus flytrap (Dionaea muscipula) after digestion of (13) C/(15) N-labelled prey.

    PubMed

    Kruse, J; Gao, P; Eibelmeier, M; Alfarraj, S; Rennenberg, H

    2017-07-20

    Amino acids represent an important component in the diet of the Venus flytrap (Dionaea muscipula), and supply plants with much needed nitrogen resources upon capture of insect prey. Little is known about the significance of prey-derived carbon backbones of amino acids for the success of Dionaea's carnivorous life-style. The present study aimed at characterizing the metabolic fate of (15) N and (13) C in amino acids acquired from double-labeled insect powder. We tracked changes in plant amino acid pools and their δ(13) C- and δ(15) N-signatures over a period of five weeks after feeding, as affected by contrasting feeding intensity and tissue type (i.e., fed and non-fed traps and attached petioles of Dionaea). Isotope signatures (i.e., δ(13) C and δ(15) N) of plant amino acid pools were strongly correlated, explaining 60% of observed variation. Residual variation was related to contrasting effects of tissue type, feeding intensity and elapsed time since feeding. Synthesis of nitrogen-rich transport compounds (i.e., amides) during peak time of prey digestion increased (15) N- relative to (13) C- abundances in amino acid pools. After completion of prey digestion, (13) C in amino acid pools was progressively exchanged for newly fixed (12) C. The latter process was most evident for non-fed traps and attached petioles of plants that had received ample insect powder. We argue that prey-derived amino acids contribute to respiratory energy gain and loss of (13) CO2 during conversion into transport compounds (i.e., 2 days after feeding), and that amino-nitrogen helps boost photosynthetic carbon gain later on (i.e., 5 weeks after feeding). © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

  6. Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.

    PubMed

    Brereton, Nicholas J B; Pitre, Frederic E; Shield, Ian; Hanley, Steven J; Ray, Michael J; Murphy, Richard J; Karp, Angela

    2014-11-01

    Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production.

  7. Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow

    PubMed Central

    Brereton, Nicholas J.B.; Pitre, Frederic E.; Shield, Ian; Hanley, Steven J.; Ray, Michael J.; Murphy, Richard J.; Karp, Angela

    2014-01-01

    Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and 15N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2–3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production. PMID:24186940

  8. Estimation of internal and external nitrogen for corals with a long-term 15N-labelling experiment and subsequent model calculations

    NASA Astrophysics Data System (ADS)

    Tanaka, Yasuaki; Grottoli, Andréa; Matsui, Yohei; Suzuki, Atsushi; Sakai, Kazuhiko

    2014-05-01

    Coral reef ecosystems maintain high primary productivity though the seawater is extremely oligotrophic. One of the hypotheses to explain this paradox is the recycling of nutrients in animal-algal symbiotic organisms such as corals. It is relatively easy to measure nutrient uptake rates by corals from seawater, but the proportion of internally circulating nutrients between the coral host and the endosymbiotic algae (zooxanthellae) is more challenging. Here, we performed a long-term and continuous 15N-labelling experiment to quantify the proportionate contribution of seawater (external N source) and the animal host (internal N source) to the total N influx in the endosymbiotic algae. Branches from the scleractinian corals Porites cylindrica and Montipora digitata from Okinawa, Japan, were cultured for 2 months in indoor, flow-through, filtered seawater tanks with the continuous supply of 15N-labelled nitrate. At the initial and after 2, 4, and 9 weeks of the study, coral branches were collected and the algal and animal fractions were separated for isotopic analyses. In both corals, the N isotope ratio of symbiotic algae exponentially increased and the values were much higher than those of the host tissue, suggesting that the algae had a faster turnover N time than the animal host. Algal and host N biomass normalized to the coral surface area slowly decreased in both coral species over the study period. To calculate the contribution of internal and external N, a simple mixing model of algal N metabolism was designed. Using differential equations of 15N balance and N biomass balance, F1 and F2 (external and internal N fluxes to symbiotic algae, respectively) were expressed as the functions of time. The model calculations showed that F2 was much higher than F1 in P. cylindrica and the percentage of internal N to the total influx N (PIN) was >70%. On the other hand, the contribution of F1 and F2 was comparable in M. digitata and the PIN was 40-70%. These results

  9. Studies on the solution conformation of human thioredoxin using heteronuclear sup 15 N- sup 1 H nuclear magnetic resonance spectroscopy

    SciTech Connect

    Forman-Kay, J.D. Yale Univ., New Haven, CT ); Gronenborn, A.M.; Kay, L.E.; Clore, G.M. ); Wingfield, P.T. )

    1990-02-13

    The solution conformation of uniformly labeled {sup 15}N human thioredoxin has been studied by two-dimensional heteronuclear {sup 15}N-{sup 1}H nuclear magnetic resonance spectroscopy. Assignments of the {sup 15}N resonances of the protein are obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC-correlated (COSY), and relayed HMQC-nuclear Overhauser (NOESY) spectroscopy. Values of the {sup 3}J{sub HN{alpha}} splittings for 87 of the 105 residues of thioredoxin are extracted from a variant of the HMQC-COSY experiment, known as HMQC-J, and analyzed to give accurate {sup 3}J{sub HN{alpha}} coupling constants. In addition, long-range C{sub {alpha}}H(i)-{sup 15}N(i+1) scalar connectivities are identified by heteronuclear multiple bond correlation (HMBC) spectroscopy. The presence of these three-bond scalar connectivities in predominantly {alpha}-helical regions correlates well with the secondary structure determined previously from a qualitative analysis of homonuclear nuclear Overhauser data suggesting that this technique may provide additional information for secondary structure determination a priori. The accuracy with which {sup 3}J{sub HN{alpha}} coupling constants can be obtained from the HMQC-J experiment permits a more precise delineation of the beginnings and ends of secondary structural elements of human thioredoxin and of irregularities in these elements.

  10. ENDOR and ESEEM of the 15N labelled radical cations of chlorophyll a and the primary donor P 700 in photosystem I

    NASA Astrophysics Data System (ADS)

    Käβ, H.; Bittersmann-Weidlich, E.; Andréasson, L.-E.; Bönigk, B.; Lubitz, W.

    1995-05-01

    The hyperfine couplings of the nitrogen nuclei in the radical cations of both 15N-labelled chlorophyll a and the primary donor P 700 in Photosystem I of Synechococcus elongatus and spinach ( Spinacea oleracea) in frozen solutions were investigated by ENDOR and, for confirmation, by two-dimensional ESEEM techniques. In addition, 1H ENDOR experiments were performed on these compounds. The experimental 15N hyperfine couplings of the chlorophyll a radical cation are compared with theoretical ones obtained by RHF-INDO/SP calculations and with the respective hyperfine couplings in the closely related 15N-bacteriochlorophyll a radical cation. Based on the observed 15N and 1H hyperfine couplings two possible models are discussed for P 700+: (a) the special pair model with a strongly asymmetric spin density distribution over the dimer halves; (b) the model of a strongly perturbed chlorophyll a monomer.

  11. 15N-labeling experiments to dissect the contributions of heterotrophic denitrification and anammox to nitrogen removal in the OMZ waters of the ocean.

    PubMed

    Holtappels, Moritz; Lavik, Gaute; Jensen, Marlene M; Kuypers, Marcel M M

    2011-01-01

    In recent years, (15)N-labeling experiments have become a powerful tool investigating rates and regulations of microbially mediated nitrogen loss processes in the ocean. This chapter introduces the theoretical and practical aspects of (15)N-labeling experiments to dissect the contribution of denitrification and anammox to nitrogen removal in oxygen minimum zones (OMZs). We provide a detailed description of the preparation and realization of the experiments on board. Subsequent measurements of N(2) isotopes using gas chromatography mass spectrometry as well as processing of data and calculation of anammox and denitrification rates are explained. Important supplementary measurements are specified, such as the measurement of nanomolar concentrations of ammonium, nitrite, and nitrate. Nutrient profiles and (15)N-experiments from the Peruvian OMZ are presented and discussed as an example. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Metabolic labeling with stable isotope nitrogen (15N) to follow amino acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii

    PubMed Central

    2014-01-01

    Background The length of time that a protein remains available to perform its function is significantly influenced by its turnover rate. Knowing the turnover rate of proteins involved in different processes is important to determining how long a function might progress even when the stimulus has been removed and no further synthesis of the particular proteins occurs. In this article, we describe the use of 15N-metabolic labeling coupled to GC-MS to follow the turnover of free amino acids and LC-MS/MS to identify and LC-MS to follow the turnover of specific proteins in Chlamydomonas reinhardtii. Results To achieve the metabolic labeling, the growth medium was formulated with standard Tris acetate phosphate medium (TAP) in which14NH4Cl was replaced with 15NH415NO3 and (14NH4)6Mo7O24.4H2O was replaced with Na2MoO4.2H2O. This medium designated 15N-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful 15N incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from 14N- and 15N-labeled crude algal protein extracts followed by LC-MS/MS resulted in the identification of 27 proteins. Of these, five displayed peptide sequence confidence levels greater than 95% and protein sequence coverage greater than 25%. These proteins were the RuBisCo large subunit, ATP synthase CF1 alpha and beta subunits, the mitochondrial protein (F1F0 ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized 15N-labeled free amino acids and proteins obtained maximum incorporation of the 15N-label, turnover rates were determined after transfer of cells into 14N-TAP medium. The t½ values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the 15N-fractional abundance over time. Conclusion We describe a more

  13. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

    PubMed Central

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian

    2015-01-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly 13C/15N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive 13C/15N-labeled amino acids. The most cost-effective production of 13C/15N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% 13C-glycerol and 0.5% 15N-ammonium sulfate, supplemented with only 0.025% of 13C/15N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state. PMID:25795666

  14. Synthesis of 14N and 15N-labeled trityl-nitroxide biradicals with strong spin-spin interaction and improved sensitivity to redox status and oxygen

    PubMed Central

    Liu, Yangping; Villamena, Frederick A.; Song, Yuguang; Sun, Jian; Rockenbauer, Antal

    2014-01-01

    Simultaneous evaluation redox status and oxygenation in biological systems is of great importance for the understanding of biological functions. Electron paramagnetic resonance spectroscopy coupled with the use of the nitroxide radicals have been an indispensable technique for this application but are still limited by its low oxygen sensitivity, and low EPR resolution in part due to the moderately broad EPR triplet and spin quenching through bioreduction. In this study, we showed that these drawbacks can be overcome through the use of trityl-nitroxide biradicals allowing for the simultaneous measurement of redox status and oxygenation. A new trityl-nitroxide biradical TNN14 composed of a pyrrolidinyl-nitroxide and a trityl, and its isotopically labeled 15N analogue TNN15 were synthesized and characterized. Both biradicals exhibited much stronger spin-spin interaction with J > 400 G than the previous synthesized trityl-nitroxide biradicals TN1 (~160 G) and TN2 (~52 G) with longer linker chain length. The enhanced stability of TNN14 was evaluated using ascorbate as reductant and the effect of different types of cyclodextrins on its stability in the presence of ascorbate was also investigated. Both biradicals are sensitive to redox status, and their corresponding trityl-hydroxylamines resulting from the reduction of the biradicals by ascorbate share the same oxygen sensitivity. Of note is that the 15N-labeled TNN15-H with an EPR doublet exhibits improved EPR signal amplitude as compared to TNN14-H with an EPR triplet. In addition, cyclic voltammetric studies verify the characteristic electrochemical behaviors of the trityl-nitroxide biradicals. PMID:21028905

  15. NMR study of the metabolic 15N isotopic enrichment of cyanophycin synthesized by the cyanobacterium Synechocystis sp. strain PCC 6308.

    PubMed

    Suarez, C; Kohler, S J; Allen, M M; Kolodny, N H

    1999-02-02

    1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy has been used to characterize cyanophycin, a multi-l-arginyl-poly-[l-aspartic acid] polypeptide from the cyanobacterium Synechocystis sp. strain PCC 6308. 1H, 13C and 15N chemical shifts and 1JHN and 1JCN coupling constants were measured in isolated 15N-labeled cyanophycin, and showed chemical shift values and J-couplings consistent with the reported polypeptide structure. 15N enrichment levels were determined from the extent of 1H-15N J-coupling in 1H NMR spectra of cyanophycin. Similar experiments using 13C-15N coupling in 13C NMR spectra were not useful in determining enrichment levels.

  16. New Multidimensional Editing Experiments for Measurement of Amide Deuterium Isotope Effects on C βChemical Shifts in 13C, 15N-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Meissner, Axel; Sørensen, Ole Winneche

    1998-12-01

    Novel multidimensional NMR pulse sequences for measurement of the three- and four-bond amide deuterium isotope effect on the chemical shifts of13Cβin proteins are presented. The sequences result in editing into two subspectra of a heteronuclear triple resonance spectrum {ω(N), ω(Cβ), ω(Hα)} according to there being a deuterium or a proton attached to15N for the pertinent correlations. The new experiments are demonstrated by an application to the first module of the13C,15N-labeled protein RAP 18-112 (N-terminal module of α2-macroglobulin receptor associated protein).

  17. Evaluation of 2D-ESEEM data of 15N-labeled radical cations of the primary donor P 700 in photosystem I and chlorophyll a

    NASA Astrophysics Data System (ADS)

    Käβ, H.; Lubitz, W.

    1996-03-01

    Hyperfine couplings (hfc's) of the nitrogen nuclei in the 15N-labeled radical cations of chlorophyll a and the primary donor P 700 in photosystem I of spinach were investigated in frozen solution by two-dimensional stimulated echo ESEEM. 15N hfc tensors were evaluated by comparison of the experimental data with simulations of the time domain and frequency domain ESEEM signals. The results are discussed in the framework of a chlorophyll a dimer model for the radical cation of P 700.

  18. Proteome turnover in the green alga Ostreococcus tauri by time course 15N metabolic labeling mass spectrometry.

    PubMed

    Martin, Sarah F; Munagapati, Vijaya S; Salvo-Chirnside, Eliane; Kerr, Lorraine E; Le Bihan, Thierry

    2012-01-01

    Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters; however, recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast, and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with (15)N. We applied it in a study of the unicellular pico-alga Ostreococcus tauri and observed high relative turnover in chloroplast-encoded ATPases (0.42-0.58% h(-1)), core photosystem II proteins (0.34-0.51% h(-1)), and RbcL (0.47% h(-1)), while nuclear-encoded RbcS2 is more stable (0.23% h(-1)). Mitochondrial targeted ATPases (0.14-0.16% h(-1)), photosystem antennae (0.09-0.14% h(-1)), and histones (0.07-0.1% h(-1)) were comparatively stable. The calculation of degradation and synthesis rate constants k(deg) and k(syn) confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144 h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.

  19. Uniform {sup 15}N- and {sup 15}N/{sup 13}C-labeling of proteins in mammalian cells and solution structure of the amino terminal fragment of u-PA

    SciTech Connect

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W.

    1994-12-01

    Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

  20. Biosynthesis of pyrrolnitrin. Incorporation of 13C, 15N double-labelled D- and L-tryptophan.

    PubMed

    Zhou, P; Mocek, U; Siesel, B; Floss, H G

    1992-01-01

    Experiments on the incorporation of D- and L-[alanine-3-13C,2-15N]tryptophan into the antibiotic pyrrolnitrin in Pseudomonas aureofaciens confirmed earlier conclusions about the conversion of L-tryptophan into pyrrolnitrin. They also demonstrated that a fraction of the D isomer is incorporated without breakage of the 15N-carbon bond, consistent with the operation of a second pathway from D-tryptophan to pyrrolnitrin. Cell-free experiments confirmed the conversion of 3-(o-aminophenyl)pyrrole into aminopyrrolnitrin but failed to detect enzymatic oxidation of the latter to pyrrolnitrin.

  1. Benchmark Theoretical and Experimental Study on (15)N NMR Shifts of Oxidatively Damaged Guanine.

    PubMed

    Dračínský, Martin; Šála, Michal; Klepetářová, Blanka; Šebera, Jakub; Fukal, Jiří; Holečková, Veronika; Tanaka, Yoshiyuki; Nencka, Radim; Sychrovský, Vladimír

    2016-02-11

    The (15)N NMR shifts of 9-ethyl-8-oxoguanine (OG) were calculated and measured in liquid DMSO and in crystal. The OG molecule is a model for oxidatively damaged 2'-deoxyguanosine that occurs owing to oxidative stress in cell. The DNA lesion is repaired with human 8-oxoguanine glycosylase 1 (hOGG1) base-excision repair enzyme, however, the exact mechanism of excision of damaged nucleobase with hOGG1 is currently unknown. This benchmark study on (15)N NMR shifts of OG aims their accurate structural interpretation and calibration of the calculation protocol utilizable in future studies on mechanism of hOGG1 enzyme. The effects of NMR reference, DFT functional, basis set, solvent, structure, and dynamics on calculated (15)N NMR shifts were first evaluated for OG in crystal to calibrate the best performing calculation method. The effect of large-amplitude motions on (15)N NMR shifts of OG in liquid was calculated employing molecular dynamics. The B3LYP method with Iglo-III basis used for B3LYP optimized geometry with 6-311++G(d,p) basis and including effects of solvent and molecular dynamic was the calculation protocol used for calculation of (15)N NMR shifts of OG. The NMR shift of N9 nitrogen of OG was particularly studied because the atom is involved in an N-glycosidic bond that is cleaved with hOGG1. The change of N9 NMR shift owing to oxidation of 9-ethylguanine (G) measured in liquid was -27.1 ppm. The calculated N9 NMR shift of OG deviated from experiment in crystal and in liquid by 0.45 and 0.65 ppm, respectively. The calculated change of N9 NMR shift owing to notable N9-pyramidalization of OG in one previously found polymorph was 20.53 ppm. We therefore assume that the pyramidal geometry of N9 nitrogen that could occur for damaged DNA within hOGG1 catalytic site might be detectable with (15)N NMR spectroscopy. The calculation protocol can be used for accurate structural interpretation of (15)N NMR shifts of oxidatively damaged guanine DNA residue.

  2. Assignments of the Pfr-Pr FTIR difference spectrum of cyanobacterial phytochrome Cph1 using 15N and 13C isotopically labeled phycocyanobilin chromophore.

    PubMed

    van Thor, Jasper J; Fisher, Nicholas; Rich, Peter R

    2005-11-03

    The reversible red and far-red light-induced transitions of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 were investigated by Fourier transform infrared (FTIR) difference spectroscopy. High-quality light-induced Pfr-Pr difference FTIR spectra were recorded for the 58 kDa N-terminal domain of Cph1 by repetitive photochemical cycling and signal averaging. The Pfr-Pr difference spectra in H(2)O and D(2)O were very similar to those previously reported for full-length 85 kDa Cph1.(1) Published assignments were extended by analysis of the effects of (13)C and (15)N isotope substitutions at selected sites in the phycocyanobilin chromophore and by (15)N global labeling of the protein. The Pfr-Pr difference spectra were dominated by an amide I peak/trough at 1653 cm(-1)(+)/1631 cm(-1)(-) and a smaller amide II band at 1554 cm(-1). Labeling effects allowed specific chromophore assignments for the C(1)=O (1736 cm(-1)(-)/1724 cm(-1)(+)) and C(19)=O (1704 cm(-1)(-)) carbonyl vibrations, C=C vibrations at 1589 cm(-1)(+), and bands at 1537(-), 1512(+), 1491(-), 1163(+), 1151(-), 1134(+), 1109(-), and 1072(-) cm(-1) that must involve chromophore C-N bonds. A variety of additional changes were insensitive to isotope labeling of the chromophore. Effects of (15)N labeling of the protein were used to tentatively assign some of these to specific amino acid changes. Those insensitive to (15)N labeling included a protonated aspartic or glutamic acid at 1734 cm(-1)(-)/1722 cm(-1)(+) and a cysteine at 2575 cm(-1)(+)/2557 cm(-1)(-). Bands sensitive to (15)N protein labeling at 1487 cm(-1)(+)/1502 cm(-1)(-) might arise from trytophan and bands at 1261 cm(-1)(+)/1244 cm(-1)(-) and 1107 cm(-1)(-)/1095 cm(-1)(+) might arise from a histidine environment or protonation change. These assignments are discussed in light of the 15Z-E photoisomerization model of phototransformation and the associated protein conformational changes.

  3. In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by 15N Nuclear Magnetic Resonance

    PubMed Central

    Tesch, M.; de Graaf, A. A.; Sahm, H.

    1999-01-01

    Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type. PMID:10049869

  4. Dynamic changes of the Caenorhabditis elegans proteome during ontogenesis assessed by quantitative analysis with 15N metabolic labeling.

    PubMed

    Geillinger, Kerstin E; Kuhlmann, Katja; Eisenacher, Martin; Meyer, Helmut E; Daniel, Hannelore; Spanier, Britta

    2012-09-07

    The development of the nematode Caenorhabditis elegans is a highly dynamic process. Although various studies have assessed global transcriptome changes, information on the dynamics of the proteome during ontogenesis is not available. We metabolically labeled C. elegans by using ¹⁵N ammonium chloride as a precursor in Escherichia coli feeding bacteria grown in minimal media as a new cost-effective technique. Quantitative proteome analysis was performed by LC-MS/MS in animals harvested at different times during ontogenesis. We identified and quantified 245 proteins at all larval stages in two independent replicates. Between larval stages (20 and 40 h after hatching) 61 were found to change significantly in level. Among those ribosomal proteins, aminoacyl tRNA synthetases and enzymes of energy metabolism increased in abundance, while extracellular matrix proteins and muscle proteins dominated groups displaying reduced levels. Moreover, changes observed for selected proteins such as VIT-6 and SOD-1 matched with previously published findings confirming the validity of our approach. The metabolic labeling technique applied seems well suited to assess changes in the proteome changes of C. elegans in a quantitative manner during larval development. The data set generated provides the basis for further exploitation of the role of individual proteins or protein clusters during ontogenesis.

  5. HCN, a triple-resonance NMR technique for selective observation of histidine and tryptophan side chains in 13C/15N-labeled proteins.

    PubMed

    Sudmeier, J L; Ash, E L; Günther, U L; Luo, X; Bullock, P A; Bachovchin, W W

    1996-12-01

    HCN, a new 3D NMR technique for stepwise coherence transfer from 1H to 13C to 15N and reverse through direct spin couplings 1JCH and 1JCN, is presented as a method for detection and assignment of histidine and tryptophan side-chain 1H, 13C, and 15N resonances in uniformly 13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay tau 3 were employed for determination of optimal tau 3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD) at pH 6, H(C)N, the 1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-defined cross peaks in 20 h for the 13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 12 1H and 13C chemical shifts and 10 of the 12 15N chemical shifts were determined. The 13C dimension proved essential in assignment of the multiply overlapping 1H and 15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mM sample of phenylmethanesulfonyl fluoride (PMSF)-inhibited alpha-lytic protease (MW = 19.8 kD) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited alpha-lytic protease after 18 h at various temperatures ranging from 5 to 55 degrees C, probably due to efficient relaxation of active-site imidazole 1H and/or 15N nuclei.

  6. Nitrogen-15 labeled 5S RNA. Identification of uridine base pairs in Escherichia coli 5S RNA by sup 1 H- sup 15 N multiple quantum NMR

    SciTech Connect

    Davis, D.R.; Yamaizumi, Z.; Nishimura, S.; Poulter, C.D. )

    1989-05-02

    Escherichia coli 5S RNA labeled with {sup 15}N at N3 of the uridines was isolated from the S{phi}-187 uracil auxotroph grown on a minimal medium supplemented with (3-{sup 15}N)uracil. {sup 1}H-{sup 15}N multiple quantum filtered and 2D chemical shift correlated spectra gave resonances for the uridine imino {sup 1}H-{sup 15}N units whose protons were exchanging slowly with solvent. Peaks with {sup 1}H/{sup 15}N shifts at 11.6/154.8, 11.7/155.0, 11.8/155.5, 12.1/155.0, and 12.2/155.0 ppm were assigned to GU interactions. Two labile high-field AU resonances at 12.6/156.8 and 12.8/157.3 ppm typical of Au pairs in a shielded environment at the end of a helix were seen. Intense AU signals were also found at 13.4/158.5 and 13.6/159.2 ppm where {sup 1}H-{sup 15}N units in normal Watson-Crick pairs resonate. {sup 1}H resonances at 10.6 and 13.8 ppm were too weak, presumably because of exchange with water, to give peaks in chemical shift correlated spectra. {sup 1}H chemical shifts suggest that the resonance at 13.8 ppm represents a labile AU pair, while the resonance at 10.6 ppm is typical of a tertiary interaction between U and a tightly bound water or a phosphate residue. The NMR data are consistent with proposed secondary structures for 5S RNA.

  7. Use of a novel nitrification inhibitor to reduce nitrous oxide emission from (15)N-labelled dairy slurry injected into soil.

    PubMed

    Dittert, K; Bol, R; King, R; Chadwick, D; Hatch, D

    2001-01-01

    Recent recommendations for environmentally sound use of liquid animal manure often include injection of slurry into soil. Two of the most important undesired side effects, ammonia (NH(3)) volatilisation and odour emissions, are usually significantly reduced by slurry injection. On the other hand, because of the higher amount of nitrogen (N) remaining in soil, the risk of nitrate (NO(3)(-)) leaching and nitrous oxide (N(2)O) emissions is increased. Thus, the reduction of local effects caused by NH(3) deposition, e.g. N enrichment and soil acidification, may be at the cost of large-scale effects such as ozone depletion and global warming as a result of emitted N(2)O. In this context, nitrification inhibitors can contribute significantly to a reduction in NO(3)(-) leaching and N(2)O production. A field experiment was carried out at IGER, North Wyke, which aimed to evaluate the effect of the new nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP/ENTEC). For this experiment, (15)N enriched dairy slurry was used and the isotopic label in soil N as well as in N(2)O were studied. After slurry injection into the grassland soil in August 2000, the major emissions of N(2)O occurred during the first ten days. As expected, high N(2)O emission rates and (15)N content of the emissions were concentrated on the slurry injection slots, showing a steep decrease towards the untreated centre-point between slurry injection slots. The nitrification inhibitor DMPP proved to be very efficient in reducing N(2)O emissions. At a rate of 2 kg DMPP ha(-1), the total amount of N(2)O emitted was reduced by 32%, when compared with slurry injection without DMPP. The isotopic label of the emitted N(2)O showed that during the 22-day experimental period, emissions from the slurry N pool were strongly reduced by DMPP from 0.93 kg N(2)O-N ha(-1) (-DMPP) to 0.50 kg N(2)O-N ha(-1) (+DMPP), while only a minor effect on emissions from the soil N pool was observed (0.69 to 0.60 kg N(2)O-N ha(-1

  8. HCN, A Triple-Resonance NMR Technique for Selective Observation of Histidine and Tryptophan Side Chains in 13C/ 15N-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Sudmeier, James L.; Ash, Elissa L.; Günther, Ulrich L.; Luo, Xuelian; Bullock, Peter A.; Bachovchin, William W.

    1996-12-01

    HCN, a new 3D NMR technique for stepwise coherence transfer from1H to13C to15N and reverse through direct spin couplings1JCHand1JCN, is presented as a method for detection and assignment of histidine and tryptophan side-chain1H,13C, and15N resonances in uniformly13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay τ3were employed for determination of optimal τ3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD) at pH 6, H(C)N, the1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-defined cross peaks in 20 h for the13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 121H and13C chemical shifts and 10 of the 1215N chemical shifts were determined. The13C dimension proved essential in assignment of the multiply overlapping1H and15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mMsample of phenylmethanesulfonyl fluoride (PMSF)-inhibited α-lytic protease (MW = 19.8 kD) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited α-lytic protease after 18 h at various temperatures ranging from 5 to 55°C, probably due to efficient relaxation of active-site imidazole1H and/or15N nuclei.

  9. Detection of Staphylococcus aureus using 15N-labeled bacteriophage amplification coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

    PubMed

    Pierce, Carrie L; Rees, Jon C; Fernández, Facundo M; Barr, John R

    2011-03-15

    A novel approach to rapid bacterial detection using an isotopically labeled (15)N bacteriophage and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is introduced. Current phage amplification detection (PAD) via mass spectrometric analysis is limited because host bacteria must be inoculated with low phage titers in such a way that initial infecting phage concentrations must be below the detection limit of the instrument, thus lengthening incubation times. Additionally, PAD techniques cannot distinguish inoculate input phage from output phage which can increase the possibility of false positive results. Here, we report a rapid and accurate PAD approach for identification of Staphylococcus aureus via detection of bacteriophage capsid proteins. This approach uses both a wild-type (14)N and a (15)N-isotopically labeled S. aureus-specific bacteriophage. High (15)N phage titers, above our instrument's detection limits, were used to inoculate S. aureus. MALDI-TOF MS detection of the (14)N progeny capsid proteins in the phage-amplified culture indicated the presence of the host bacteria. Successful phage amplification was observed after 90 min of incubation. The amplification was observed by both MALDI-TOF MS analysis and by standard plaque assay measurements. This method overcomes current limitations by improving analysis times while increasing selectivity when compared to previously reported PAD methodologies.

  10. Efficient Measurement of 3JN,Cγ and 3JC‧,Cγ Coupling Constants of Aromatic Residues in 13C, 15N-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Löhr, Frank; Rüterjans, Heinz

    2000-09-01

    An NMR pulse sequence is proposed for the simultaneous determination of side chain χ1 torsion-angle related 3JN,Cγ and 3JC‧,Cγ couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated 15N and 1H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly 13C/15N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.

  11. Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry.

    PubMed

    Giavalisco, Patrick; Li, Yan; Matthes, Annemarie; Eckhardt, Aenne; Hubberten, Hans-Michael; Hesse, Holger; Segu, Shruthi; Hummel, Jan; Köhl, Karin; Willmitzer, Lothar

    2011-10-01

    The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.

  12. 13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans

    PubMed Central

    Drechsler, Robin; Gafken, Philip R.; Olsen, Carissa Perez

    2015-01-01

    Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining 13C and 15N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism. PMID:26528916

  13. UV-visible and (1)H-(15)N NMR spectroscopic studies of colorimetric thiosemicarbazide anion sensors.

    PubMed

    Farrugia, Kristina N; Makuc, Damjan; Podborska, Agnieszka; Szaciłowski, Konrad; Plavec, Janez; Magri, David C

    2015-02-14

    Four model thiosemicarbazide anion chemosensors containing three N-H bonds, substituted with phenyl and/or 4-nitrophenyl units, were synthesised and studied for their anion binding abilities with hydroxide, fluoride, acetate, dihydrogen phosphate and chloride. The anion binding properties were studied in DMSO and 9 : 1 DMSO-H2O by UV-visible absorption and (1)H/(13)C/(15)N NMR spectroscopic techniques and corroborated with DFT studies. Significant changes were observed in the UV-visible absorption spectra with all anions, except for chloride, accompanied by dramatic colour changes visible to the naked eye. These changes were determined to be due to the deprotonation of the central N-H proton and not due to hydrogen bonding based on (1)H/(15)N NMR titration studies with acetate in DMSO-d6-0.5% water. Direct evidence for deprotonation was confirmed by the disappearance of the central thiourea proton and the formation of acetic acid. DFT and charge distribution calculations suggest that for all four compounds the central N-H proton is the most acidic. Hence, the anion chemosensors operate by a deprotonation mechanism of the central N-H proton rather than by hydrogen bonding as is often reported.

  14. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant tissue isotope labeling

    USDA-ARS?s Scientific Manuscript database

    Tracing heavy stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O o...

  15. Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots*

    PubMed Central

    Li, Lei; Nelson, Clark J.; Solheim, Cory; Whelan, James; Millar, A. Harvey

    2012-01-01

    The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a 15N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (KS) and degradation (KD) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify KS and KD for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. KS and KD correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive 15N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability. PMID:22215636

  16. Selecting matched root architecture in tree pairs to be used for assessing N 2 fixation based on soil- 15N-labelling

    NASA Astrophysics Data System (ADS)

    Nasr, Hafedh; Ghorbel, Mohamed Habib; Wallander, Håkan; Dommergues, Yvon René

    2005-03-01

    It is commonly assumed that soil- 15N-labelling provides reliable estimates of N 2 fixation in trees by matching N 2-fixing and non-N 2-fixing tree pairs. As root system is a key parameter in determining suitability of the tree pairs, we compared root architecture of Acacia cyanophylla Lindl. and Casuarina glauca Sieber ex. Spreng. (two N 2-fixing trees) with Eucalyptus camaldulensis Dehn. and Ceratonia siliqua L. (two non-N 2-fixing trees) at 4-year-old in Mediterranean-semiarid zone. The rhizobium strain used appeared more motile than Frankia strain. A. cyanophylla and E. camaldulensis had extensive rooting area and volume of fine roots, and both species tended to develop marked horizontal rooting, compared to C. glauca and C. siliqua. Characteristics of fine- and horizontal-root components can be used in selecting matched root systems of N 2-fixing and reference-paired trees. Root architecture of C. glauca was more similar to C. siliqua, than to E. camaldulensis, and that of A. cyanophylla was more similar to E. camaldulensis than to C. siliqua. Accordingly, E. camaldulensis is an appropriate reference to estimate actual N 2 fixation by A. cyanophylla, and C. siliqua is an appropriate reference for C. glauca, when using soil- 15N-labelling method in the prevailing site environment.

  17. Determining degradation and synthesis rates of arabidopsis proteins using the kinetics of progressive 15N labeling of two-dimensional gel-separated protein spots.

    PubMed

    Li, Lei; Nelson, Clark J; Solheim, Cory; Whelan, James; Millar, A Harvey

    2012-06-01

    The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.

  18. Biosynthesis of 15N-labeled cylindrospermopsin and its application as internal standard in stable isotope dilution analysis.

    PubMed

    Kittler, Katrin; Hoffmann, Holger; Lindemann, Franziska; Koch, Matthias; Rohn, Sascha; Maul, Ronald

    2014-09-01

    Cylindrospermopsin (CYN) is a cyanobacterial toxin associated with human and animal poisonings. Due to its toxicity in combination with its widespread occurrence, the development of reliable methods for selective, sensitive detection and accurate quantification is mandatory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using stable isotope dilution analysis (SIDA) represents an ideal tool for this purpose. U-[(15)N5]-CYN was synthesized by culturing Aphanizomenon flos-aquae in Na(15)NO3-containing cyanobacteria growth medium followed by a cleanup using graphitized carbon black columns and mass spectrometric characterization. Subsequently, a SIDA-LC-MS/MS method for the quantification of CYN in freshwater and Brassica matrices was developed showing satisfactory performance data. The recovery ranged between 98 and 103 %; the limit of quantification was 15 ng/L in freshwater and 50 μg/kg dry weight in Brassica samples. The novel SIDA was applied for CYN determination in real freshwater samples as well as in kale and in vegetable mustard exposed to toxin-containing irrigation water. Two of the freshwater samples taken from German lakes were found to be CYN-contaminated above limit of quantification (17.9 and 60.8 ng/L). CYN is systemically available to the examined vegetable species after exposure of the rootstock leading to CYN mass fractions in kale and vegetable mustard leaves of 15.0 μg/kg fresh weight and 23.9 μg/kg fresh weight, respectively. CYN measurements in both matrices are exemplary for the versatile applicability of the developed method in environmental analysis.

  19. Balancing the (carbon) budget: Using linear inverse models to estimate carbon flows and mass-balance 13C:15N labelling experiments in low oxygen sediments.

    NASA Astrophysics Data System (ADS)

    Hunter, William Ross; Van Oevelen, Dick; Witte, Ursula

    2013-04-01

    Over 1 million km2 of seafloor experience permanent low-oxygen conditions within oxygen minimum zones (OMZs). OMZs are predicted to grow as a consequence of climate change, potentially affecting oceanic biogeochemical cycles. The Arabian Sea OMZ impinges upon the western Indian continental margin at bathyal depths (150 - 1500m) producing a strong depth dependent oxygen gradient at the sea floor. The influence of the OMZ upon the short term processing of organic matter by sediment ecosystems was investigated using in situ stable isotope pulse chase experiments. These deployed doses of 13C:15N labeled organic matter onto the sediment surface at four stations from across the OMZ (water depth 540 - 1100 m; [O2] = 0.35 - 15 μM). In order to prevent experimentally anoxia, the mesocosms were not sealed. 13C and 15N labels were traced into sediment, bacteria, fauna and 13C into sediment porewater DIC and DOC. However, the DIC and DOC flux to the water column could not be measured, limiting our capacity to obtain mass-balance for C in each experimental mesocosm. Linear Inverse Modeling (LIM) provides a method to obtain a mass-balanced model of carbon flow that integrates stable-isotope tracer data with community biomass and biogeochemical flux data from a range of sources. Here we present an adaptation of the LIM methodology used to investigate how ecosystem structure influenced carbon flow across the Indian margin OMZ. We demonstrate how oxygen conditions affect food-web complexity, affecting the linkages between the bacteria, foraminifera and metazoan fauna, and their contributions to benthic respiration. The food-web models demonstrate how changes in ecosystem complexity are associated with oxygen availability across the OMZ and allow us to obtain a complete carbon budget for the stationa where stable-isotope labelling experiments were conducted.

  20. Competition for /sup 15/N labelled ammonium between Trifolium subterraneum L. and Lolium multiflorium L. when grown in a mixture

    SciTech Connect

    Munoz Espinoza, A.E.

    1985-01-01

    Grasses and legumes are often grown in association in pastures because total herbage yield and forage quality is often higher than a monoculture grass sward. The competitive ability of the forage species for mineral N influences the stability of the mixed pasture. Mt. Barker subterranean clover (Trifolium subterraneum L.) Gulf ryegrass (Lolium multiflorium L.) were grown in pure stands and in mixtures in 3.8 I pots filled with exploded vermiculite to quantity the competition for applied mineral nitrogen (N). The isotope dilution technique using /sup 15/NH/sub 4/ was used. Also, the effect of increasing rates of fertilizer N on nodulation and fixation by subterranean clover and the rate of N uptake of subterranean clover and ryegrass was measured. The acetylene-ethylene technique and nodulation rating were used to examine the effect of N fertilization on fixation and nodulation. The uptake of N fertilizer by subterranean clover and ryegrass in both pure stands and the mixtures increased as the rate of fertilizer N applied increased. When grown in a pure stand, subterranean clover recovered a comparable amount of fertilizer N, but when grown in mixture with ryegrass, subterranean clover recovered from 44 to 364 mg of N. Ryegrass recovered 2 times the amount of labelled N in 6 hours than subterranean clover. The rate of N uptake was not due to differences in root fresh weight or dry weight. Ryegrass appeared to be a better competitor for NH/sub 4/ than subterranean clover because of the greater rate of uptake.

  1. Accessible NMR Experiments Studying the Hydrodynamics of [subscript 15]N-Enriched Ubiquitin at Low Fields

    ERIC Educational Resources Information Center

    Thompson, Laura E.; Rovnyak, David

    2007-01-01

    We have recently developed and implemented two experiments in biomolecular NMR for an undergraduate-level biophysical chemistry laboratory with commercially available [subscript 15]N-enriched human ubiquitin. These experiments take advantage of [subscript 15]N direct detection of the NMR signal. The first experiment develops skills in acquiring…

  2. Accessible NMR Experiments Studying the Hydrodynamics of [superscript 15]N-Enriched Ubiquitin at Low Fields

    ERIC Educational Resources Information Center

    Thompson, Laura E.; Rovnyak, David

    2007-01-01

    We have recently developed and implemented two experiments in biomolecular NMR for an undergraduate-level biophysical chemistry laboratory with commercially available [superscript 15]N-enriched human ubiquitin. These experiments take advantage of [superscript 15]N direct detection of the NMR signal. The first experiment develops skills in…

  3. Accessible NMR Experiments Studying the Hydrodynamics of [subscript 15]N-Enriched Ubiquitin at Low Fields

    ERIC Educational Resources Information Center

    Thompson, Laura E.; Rovnyak, David

    2007-01-01

    We have recently developed and implemented two experiments in biomolecular NMR for an undergraduate-level biophysical chemistry laboratory with commercially available [subscript 15]N-enriched human ubiquitin. These experiments take advantage of [subscript 15]N direct detection of the NMR signal. The first experiment develops skills in acquiring…

  4. Accessible NMR Experiments Studying the Hydrodynamics of [superscript 15]N-Enriched Ubiquitin at Low Fields

    ERIC Educational Resources Information Center

    Thompson, Laura E.; Rovnyak, David

    2007-01-01

    We have recently developed and implemented two experiments in biomolecular NMR for an undergraduate-level biophysical chemistry laboratory with commercially available [superscript 15]N-enriched human ubiquitin. These experiments take advantage of [superscript 15]N direct detection of the NMR signal. The first experiment develops skills in…

  5. Sinks for nitrogen inputs in terrestrial ecosystems: a meta-analysis of 15N tracer field studies

    USGS Publications Warehouse

    Templer, P.H.; Mack, M.C.; Chapin, F. S.; Christenson, L.M.; Compton, J.E.; Crook, H.D.; Currie, W.S.; Curtis, C.J.; Dail, D.B.; D'Antonio, C. M.; Emmett, B.A.; Epstein, H.E.; Goodale, C.L.; Gundersen, P.; Hobbie, S.E.; Holland, K.; Hooper, D.U.; Hungate, B.A.; Lamontagne, S.; Nadelhoffer, K.J.; Osenberg, C.W.; Perakis, S.S.; Schleppi, P.; Schimel, J.; Schmidt, I.K.; Sommerkorn, M.; Spoelstra, J.; Tietema, A.; Wessel, W.W.; Zak, D.R.

    2012-01-01

    Effects of anthropogenic nitrogen (N) deposition and the ability of terrestrial ecosystems to store carbon (C) depend in part on the amount of N retained in the system and its partitioning among plant and soil pools. We conducted a meta-analysis of studies at 48 sites across four continents that used enriched 15N isotope tracers in order to synthesize information about total ecosystem N retention (i.e., total ecosystem 15N recovery in plant and soil pools) across natural systems and N partitioning among ecosystem pools. The greatest recoveries of ecosystem 15N tracer occurred in shrublands (mean, 89.5%) and wetlands (84.8%) followed by forests (74.9%) and grasslands (51.8%). In the short term (15N tracer application), total ecosystem 15N recovery was negatively correlated with fine-root and soil 15N natural abundance, and organic soil C and N concentration but was positively correlated with mean annual temperature and mineral soil C:N. In the longer term (3–18 months after 15N tracer application), total ecosystem 15N retention was negatively correlated with foliar natural-abundance 15N but was positively correlated with mineral soil C and N concentration and C: N, showing that plant and soil natural-abundance 15N and soil C:N are good indicators of total ecosystem N retention. Foliar N concentration was not significantly related to ecosystem 15N tracer recovery, suggesting that plant N status is not a good predictor of total ecosystem N retention. Because the largest ecosystem sinks for 15N tracer were below ground in forests, shrublands, and grasslands, we conclude that growth enhancement and potential for increased C storage in aboveground biomass from atmospheric N deposition is likely to be modest in these ecosystems. Total ecosystem 15N recovery decreased with N fertilization, with an apparent threshold fertilization rate of 46 kg N·ha-1·yr-1 above which most ecosystems showed net losses of applied 15N tracer in response to N fertilizer addition.

  6. Sinks for nitrogen inputs in terrestrial ecosystems: a meta-analysis of 15N tracer field studies.

    PubMed

    Templer, P H; Mack, M C; Chapin, F S; Christenson, L M; Compton, J E; Crook, H D; Currie, W S; Curtis, C J; Dail, D B; D'Antonio, C M; Emmett, B A; Epstein, H E; Goodale, C L; Gundersen, P; Hobbie, S E; Holland, K; Hooper, D U; Hungate, B A; Lamontagne, S; Nadelhoffer, K J; Osenberg, C W; Perakis, S S; Schleppi, P; Schimel, J; Schmidt, I K; Sommerkorn, M; Spoelstra, J; Tietema, A; Wessel, W W; Zak, D R

    2012-08-01

    Effects of anthropogenic nitrogen (N) deposition and the ability of terrestrial ecosystems to store carbon (C) depend in part on the amount of N retained in the system and its partitioning among plant and soil pools. We conducted a meta-analysis of studies at 48 sites across four continents that used enriched 15N isotope tracers in order to synthesize information about total ecosystem N retention (i.e., total ecosystem 15N recovery in plant and soil pools) across natural systems and N partitioning among ecosystem pools. The greatest recoveries of ecosystem 15N tracer occurred in shrublands (mean, 89.5%) and wetlands (84.8%) followed by forests (74.9%) and grasslands (51.8%). In the short term (< 1 week after 15N tracer application), total ecosystem 15N recovery was negatively correlated with fine-root and soil 15N natural abundance, and organic soil C and N concentration but was positively correlated with mean annual temperature and mineral soil C:N. In the longer term (3-18 months after 15N tracer application), total ecosystem 15N retention was negatively correlated with foliar natural-abundance 15N but was positively correlated with mineral soil C and N concentration and C:N, showing that plant and soil natural-abundance 15N and soil C:N are good indicators of total ecosystem N retention. Foliar N concentration was not significantly related to ecosystem 15N tracer recovery, suggesting that plant N status is not a good predictor of total ecosystem N retention. Because the largest ecosystem sinks for 15N tracer were below ground in forests, shrublands, and grasslands, we conclude that growth enhancement and potential for increased C storage in aboveground biomass from atmospheric N deposition is likely to be modest in these ecosystems. Total ecosystem 15N recovery decreased with N fertilization, with an apparent threshold fertilization rate of 46 kg N x ha(-1) x yr(-1) above which most ecosystems showed net losses of applied 15N tracer in response to N fertilizer

  7. Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [13C,15N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

    PubMed Central

    2014-01-01

    Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein

  8. 2D ESEEM of the [sup 15]N-labeled radical cations of bacteriochlorophyll a and of the primary donor in reaction centers of Rhodobacter sphaeroides

    SciTech Connect

    Kaess, H.; Rautter, J.; Boenigk, B.; Lubitz, W. ); Hoefer, P. )

    1995-01-05

    The radical cations of [[sup 15]N]bacteriochlorophyll a and of the primary donor P[sub 865] in [sup 15]N-labeled reaction centers of Rhodobacter spaeroides R-26.1 were investigated in frozen solutions using two-dimensional ESEEM methods. Both three-pulse (stimulated echo) and four-pulse (HYSCORE) sequences were employed to avoid ambiguities in data analysis. Computer simulations of the experimental powder spectra were performed, yielding a complete set of nitrogen hyperfine coupling tensors for both systems. The obtained tensor values are compared with those from ENDOR measurements and from semiempirical INDO-type MO calculations. The results obtained from 2D stimulated echo ESEEM and HYSCORE for P[sub 865][sup [center dot]+] are interpreted in terms of an asymmetric spin density distribution over the halves of the bacteriochlorophyll dimer ([open quote]special pair[close quote]) in the reaction center with a ratio of approximately 5:1. This asymmetry is considerably larger at low temperatures in the frozen state than at room temperature. It is postulated that two different conformational states of the dimer exist at these temperatures with different spin density distributions. 60 refs., 9 figs., 4 tabs.

  9. Application of C30B15N15 heterofullerene in the isoniazid drug delivery: DFT studies

    NASA Astrophysics Data System (ADS)

    Hazrati, Mehrnoosh Khodam; Bagheri, Zargham; Bodaghi, Ali

    2017-05-01

    Using density functional theory, we have investigated the potential application of a C30B15N15 heterofullerene in anti-cancer isoniazid drug delivery. It was found that isoniazid prefers to attach via its -NH2 group to a boron atom of the C30B15N15 with releasing a large energy of about 21.91 kcal/mol. Our partial density of states analysis demonstrates that the boron atoms significantly contribute in generation of virtual orbitals of C30B15N15 fullerene, indicating that these atoms will be suitable for nucleophilic attack rather than carbon atoms. In addition to the large released energy, the electronic properties C30B15N15 are significantly sensitive to the isoniazid attachment which can recognize the drug trajectory by affecting the fluorescence emission properties. Unlike, different nanostructures whose structures need to be manipulated to be suitable for drug delivery, the C30B15N15 fullerene can be used in the pristine form. We proposed a drug release mechanism in cancer tissues, representing that in the low pH of the cancer cells the drug and C30B15N15 fullerene are considerably protonated, thereby separating the drug from the surface of the fullerene. The reaction mechanism of the drug with the fullerene is changed from covalence in natural environment to hydrogen bonding in acidic cancer cells.

  10. Short-term and seasonal soil nitrogen dynamics and recovery by bermudagrass irrigated with 15N labeled swine lagoon effluent

    USDA-ARS?s Scientific Manuscript database

    The experiment was conducted at a commercial swine operation located in Lowndes County, Mississippi in an acid Vaiden silty clay (very fine, montmorillonitic, thermic Vertic Hapludalf) used in this study is representative of the Alabama and Mississippi Blackland Prairie major land resource area. A ...

  11. Tracking the fate of nitrate through pulse-flow wetlands: A mesocosm scale 15N enrichment tracer study

    USGS Publications Warehouse

    Messer, Tiffany L.; Burchell, Michael R.; Böhlke, John Karl; Tobias, Craig R.

    2017-01-01

    Quantitative information about the fate of applied nitrate (NO3-N) in pulse-flow constructed wetlands is essential for designing wetland treatment systems and assessing their nitrogen removal services for agricultural and stormwater applications. Although many studies have documented NO3-N losses in wetlands, controlled experiments indicating the relative importance of different processes and N sinks are scarce. In the current study, 15NO3-N isotope enrichment tracer experiments were conducted in wetland mesocosms of two different wetland soil types at two realistic agricultural NO3-N source loads. The 15N label was traced from the source NO3-N into plant biomass, soil (including organic matter and ammonium), and N-gas constituents over 7–10 day study periods. All sinks responded positively to higher NO3-N loading. Plant uptake exceeded denitrification 2–3 fold in the low NO3-N loading experiments, while both fates were nearly equivalent in the high loading experiments. One to two years later, soils largely retained the assimilated tracer N, whereas plants had lost much of it. Results demonstrated that plant and microbial assimilation in the soil (temporary N sinks) can exceed denitrification (permanent N loss) in pulse-flow environments and must be considered by wetland designers and managers for optimizing nitrogen removal potential.

  12. Amino-acid selective experiments on uniformly 13C and 15N labeled proteins by MAS NMR: Filtering of lysines and arginines.

    PubMed

    Jehle, Stefan; Rehbein, Kristina; Diehl, Anne; van Rossum, Barth-Jan

    2006-12-01

    Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the alpha-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C-13C lysine-only correlation experiment.

  13. Qualitative study of substituent effects on NMR (15)N and (17)O chemical shifts.

    PubMed

    Contreras, Rubén H; Llorente, Tomás; Pagola, Gabriel I; Bustamante, Manuel G; Pasqualini, Enrique E; Melo, Juan I; Tormena, Cláudio F

    2009-09-10

    A qualitative approach to analyze the electronic origin of substituent effects on the paramagnetic part of chemical shifts is described and applied to few model systems, where its potentiality can be appreciated. The formulation of this approach is based on the following grounds. The influence of different inter- or intramolecular interactions on a second-order property can be qualitatively predicted if it can be known how they affect the main virtual excitations entering into that second-order property. A set of consistent approximations are introduced in order to analyze the behavior of occupied and virtual orbitals that define some experimental trends of magnetic shielding constants. This approach is applied first to study the electronic origin of methyl-beta substituent effects on both (15)N and (17)O chemical shifts, and afterward it is applied to a couple of examples of long-range substituent effects originated in charge transfer interactions such as the conjugative effect in aromatic compounds and sigma-hyperconjugative interactions in saturated multicyclic compounds.

  14. Ecotoxicity of cadmium in a soil collembolan-predatory mite food chain: Can we use the (15)N labeled litter addition method to assess soil functional change?

    PubMed

    Zhu, Dong; Ke, Xin; Wu, Longhua; Li, Zhu; Christie, Peter; Luo, Yongming

    2016-12-01

    Effects of cadmium (Cd) on predator-prey relationships and soil ecological function are poorly understood and there are few methods available to measure soil functional change. Thus, we structured a soil-dwelling food chain containing the predatory mite Hypoaspis aculeifer and its collembolan prey Folsomia candida to study the effects of Cd exposure for eight weeks in a spiked soil aged for five years. The (15)N labeled litter was added as food to analyze the change in nitrogen (N) transfer content. H. aculeifer reproduction and growth and the survival and reproduction of F. candida were all negatively affected by Cd exposure, and H. aculeifer reproduction was the most sensitive parameter. The sensitivity responses of F. candida and H. aculeifer were different from those using the previous single species test. The results suggest that predator-prey interactions might influence the toxicity of Cd by predation and food restriction. Cadmium lethal body concentrations of adults and juveniles of F. candida and H. aculeifer juveniles were 500-600, 180-270 and 8-10 μg g(-1), respectively. The content of N transfer from litter to animals in the food chain decreased significantly with increasing soil Cd concentration between 100 and 400 mg kg(-1). The results suggest that the (15)N labeled litter addition method is potentially useful for quantitative assessment of soil functional change for further risk assessment purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Natural-abundance 15N NMR studies of Turkey ovomucoid third domain. Assignment of peptide 15N resonances to the residues at the reactive site region via proton-detected multiple-quantum coherence

    NASA Astrophysics Data System (ADS)

    Ortiz-Polo, Gilberto; Krishnamoorthi, R.; Markley, John L.; Live, David H.; Davis, Donald G.; Cowburn, David

    Heteronuclear two-dimensional 1H{ 15N} multiple-quantum (MQ) spectroscopy has been applied to a protein sample at natural abundance: ovomucoid third domain from turkey ( Meleagris gallopavo), a serine proteinase inhibitor of 56 amino acid residues. Peptide amide 1H NMR assignments obtained by two-dimensional 1H{ 1H} NMR methods (R. Krishnamoorthi and J. L. Markley, unpublished data) led to identification of the corresponding 1H{ 15N} MQ coherence cross peaks. From these, 15N NMR chemical shifts were determined for several specific backbone amide groups of amino acid residues located around the reactive site region of the inhibitor. The results suggest that amide 15N chemical shifts, which are readily obtained in this way, may serve as sensitive probes for conformational studies of proteins.

  16. Dynamics of Exogenous Nitrogen Partitioning and Nitrogen Remobilization from Vegetative Organs in Pea Revealed by 15N in Vivo Labeling throughout Seed Filling1

    PubMed Central

    Schiltz, Séverine; Munier-Jolain, Nathalie; Jeudy, Christian; Burstin, Judith; Salon, Christophe

    2005-01-01

    The fluxes of (1) exogenous nitrogen (N) assimilation and (2) remobilization of endogenous N from vegetative plant compartments were measured by 15N labeling during the seed-filling period in pea (Pisum sativum L. cv Caméor), to better understand the mechanism of N remobilization. While the majority (86%) of exogenous N was allocated to the vegetative organs before the beginning of seed filling, this fraction decreased to 45% at the onset of seed filling, the remainder being directed to seeds. Nitrogen remobilization from vegetative parts contributed to 71% of the total N in mature seeds borne on the first two nodes (first stratum). The contribution of remobilized N to total seed N varied, with the highest proportion at the beginning of filling; it was independent of the developmental stage of each stratum of seeds, suggesting that remobilized N forms a unique pool, managed at the whole-plant level and supplied to all filling seeds whatever their position on the plant. Once seed filling starts, N is remobilized from all vegetative organs: 30% of the total N accumulated in seeds was remobilized from leaves, 20% from pod walls, 11% from roots, and 10% from stems. The rate of N remobilization was maximal when seeds of all the different strata were filling, consistent with regulation according to the N demand of seeds. At later stages of seed filling, the rate of remobilization decreases and may become controlled by the amount of residual N in vegetative tissues. PMID:15793068

  17. Dynamics of exogenous nitrogen partitioning and nitrogen remobilization from vegetative organs in pea revealed by 15N in vivo labeling throughout seed filling.

    PubMed

    Schiltz, Séverine; Munier-Jolain, Nathalie; Jeudy, Christian; Burstin, Judith; Salon, Christophe

    2005-04-01

    The fluxes of (1) exogenous nitrogen (N) assimilation and (2) remobilization of endogenous N from vegetative plant compartments were measured by 15N labeling during the seed-filling period in pea (Pisum sativum L. cv Cameor), to better understand the mechanism of N remobilization. While the majority (86%) of exogenous N was allocated to the vegetative organs before the beginning of seed filling, this fraction decreased to 45% at the onset of seed filling, the remainder being directed to seeds. Nitrogen remobilization from vegetative parts contributed to 71% of the total N in mature seeds borne on the first two nodes (first stratum). The contribution of remobilized N to total seed N varied, with the highest proportion at the beginning of filling; it was independent of the developmental stage of each stratum of seeds, suggesting that remobilized N forms a unique pool, managed at the whole-plant level and supplied to all filling seeds whatever their position on the plant. Once seed filling starts, N is remobilized from all vegetative organs: 30% of the total N accumulated in seeds was remobilized from leaves, 20% from pod walls, 11% from roots, and 10% from stems. The rate of N remobilization was maximal when seeds of all the different strata were filling, consistent with regulation according to the N demand of seeds. At later stages of seed filling, the rate of remobilization decreases and may become controlled by the amount of residual N in vegetative tissues.

  18. Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in (15)N-(13)C-ILV methyl labeled proteins.

    PubMed

    Frueh, Dominique P; Leed, Alison; Arthanari, Haribabu; Koglin, Alexander; Walsh, Christopher T; Wagner, Gerhard

    2009-11-01

    We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record (13)C- and (15)N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins.

  19. Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in 15N-13C-ILV methyl labeled proteins

    PubMed Central

    Frueh, Dominique P.; Leed, Alison; Arthanari, Haribabu; Koglin, Alexander; Walsh, Christopher T.; Wagner, Gerhard

    2009-01-01

    We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record 13C- and 15N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 kDa and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins. PMID:19728110

  20. Compound specific amino acid δ15N in marine sediments: A new approach for studies of the marine nitrogen cycle

    NASA Astrophysics Data System (ADS)

    Batista, Fabian C.; Ravelo, A. Christina; Crusius, John; Casso, Michael A.; McCarthy, Matthew D.

    2014-10-01

    The nitrogen (N) isotopic composition (δ15N) of bulk sedimentary N (δ15Nbulk) is a common tool for studying past biogeochemical cycling in the paleoceanographic record. Empirical evidence suggests that natural fluctuations in the δ15N of surface nutrient N are reflected in the δ15N of exported planktonic biomass and in sedimentary δ15Nbulk. However, δ15Nbulk is an analysis of total combustible sedimentary N, and therefore also includes mixtures of N sources and/or selective removal or preservation of N-containing compounds. Compound-specific nitrogen isotope analyses of individual amino acids (δ15NAA) are novel measurements with the potential to decouple δ15N changes in nutrient N from trophic effects, two main processes that can influence δ15Nbulk records. As a proof of concept study to examine how δ15NAA can be applied in marine sedimentary systems, we compare the δ15NAA signatures of surface and sinking POM sources with shallow surface sediments from the Santa Barbara Basin, a sub-oxic depositional environmental that exhibits excellent preservation of sedimentary organic matter. Our results demonstrate that δ15NAA signatures of both planktonic biomass and sinking POM are well preserved in such surface sediments. However, we also observed an unexpected inverse correlation between δ15N value of phenylalanine (δ15NPhe; the best AA proxy for N isotopic value at the base of the food web) and calculated trophic position. We used a simple N isotope mass balance model to confirm that over long time scales, δ15NPhe values should in fact be directly dependent on shifts in ecosystem trophic position. While this result may appear incongruent with current applications of δ15NAA in food webs, it is consistent with expectations that paleoarchives will integrate N dynamics over much longer timescales. We therefore propose that for paleoceanographic applications, key δ15NAA parameters are ecosystem trophic position, which determines relative partitioning of 15N

  1. NMR studies of isotopically labeled RNA

    SciTech Connect

    Pardi, A.

    1994-12-01

    In summary, the ability to generate NMR quantities of {sup 15}N and {sup 13}C-labeled RNAs has led to the development of heteronuclear multi-dimensional NMR techniques for simplifying the resonance assignment and structure determination of RNAs. These methods for synthesizing isotopically labeled RNAs are only several years old, and thus there are still relatively few applications of heteronuclear multi-dimensional NMR techniques to RNA. However, given the critical role that RNAs play in cellular function, one can expect to see an increasing number of NMR structural studies of biologically active RNAs.

  2. (13)C and (15)N solid-state NMR studies on albendazole and cyclodextrin albendazole complexes.

    PubMed

    Ferreira, M João G; García, A; Leonardi, D; Salomon, Claudio J; Lamas, M Celina; Nunes, Teresa G

    2015-06-05

    (13)C and (15)N solid-state nuclear magnetic resonance (NMR) spectra were recorded from albendazole (ABZ) and from ABZ:β-cyclodextrin, ABZ:methyl-β-cyclodextrin, ABZ:hydroxypropyl-β-cyclodextrin and ABZ:citrate-β-cyclodextrin, which were prepared by the spray-drying technique. ABZ signals were typical of a crystalline solid for the pure drug and of an amorphous compound obtained from ABZ:cyclodextrin samples. Relevant spectral differences were correlated with chemical interaction between ABZ and cyclodextrins. The number and type of complexes revealed a strong dependence on the cyclodextrin group substituent. Solid-state NMR data were consistent with the presence of stable inclusion complexes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Using natural 15N abundances to trace the fate of waste-derived nitrogen in forest ecosystems: New Zealand case studies.

    PubMed

    Wang, H; Magesan, G N; Clinton, P W; Lavery, J M

    2005-03-01

    Treatment of wastewater generally results in elevated natural 15N abundance (delta15N) in the effluent and sludges. For example, high delta15N values are found in treated sewage effluent, biosolids, and other wastes that are commonly applied to land. In contrast, N deficient coniferous forest soils usually have a low delta15N. When wastes with high delta15N values are applied to land, their distinctive delta15N signature can potentially be used to trace the fate of waste-derived N in the ecosystem. In this paper, we provide an overview of the use of delta15N in land application of wastes, including New Zealand case studies on tracing nitrogen in forest ecosystems.

  4. First detection of nitrogen from NO x in tree rings: a 15N/ 14N study near a motorway

    NASA Astrophysics Data System (ADS)

    Saurer, M.; Cherubini, P.; Ammann, M.; De Cinti, B.; Siegwolf, R.

    Nitrogen isotope analysis ( δ15N) of tree rings is potentially useful for evaluating the temporal development of the nitrogen (N) deposition to forests and for studying the long-term effects of N accumulation in ecosystems. To test this hypothesis, we investigated three sites across a pollution gradient in differing distances (20,150,1000 m) from a motorway in Switzerland, which was built in 1965. We sampled four Picea abies trees per site, whereby we extracted the tree ring cores with hot water and solvents before the isotope analysis to remove mobile N storage compounds, and determined the isotope variations in the stem wood for the period 1928-2000. While tree ring growth was not affected by the construction of the motorway, the δ15N values were increasing by up to 7.9‰ after 1965 at the most polluted site, indicating the uptake of NO x from car exhausts, although the signal was highly variable. Isotopically heavy NO x emissions were observed in an earlier study at the same location resulting in a δ15N-gradient of recent needles from +1.3‰ to -4.4‰ with increasing distance from the motorway. This gradient was also reflected in the tree rings, but dampened by a factor of about 2 compared to the needles. For the trees near the motorway, the total nitrogen concentration in the tree rings varied in parallel with the δ15N values ( r2=0.52). This enabled us to apply a mass balance equation for reconstructing the isotope signal of N originating from the car exhausts for the period 1965-2000, with the δ15N of NO 2 in the range +1.3‰-+6.4‰. The more distant sites were much less affected by the traffic and their isotope ratio reflected the influence of varying proportions of isotopically heavy (NO 2) and light (NH x) deposition. We conclude that the analysis of tree ring 15N variations is a promising tool for the detection of the role played by nitrogen deposition to the forests.

  5. Grass species influence on plant N uptake - Determination of atmospheric N deposition to a semi-natural peat bog site using a 15N labelling approach

    NASA Astrophysics Data System (ADS)

    Hurkuck, Miriam; Brümmer, Christian; Spott, Oliver; Flessa, Heinz; Kutsch, Werner L.

    2014-05-01

    Large areas of natural peat bogs in Northwestern Germany have been converted to arable land and were subjected to draining and peat cutting in the past. The few protected peatland areas remaining are affected by high nitrogen (N) deposition. Our study site - a moderately drained raised bog - is surrounded by highly fertilized agricultural land and livestock production. In this study, we used a 15N pool dilution technique called 'Integrated Total Nitrogen Input' (ITNI) to quantify annual deposition of atmospheric N into biomonitoring pots over a two-year period. Since it considers direct N uptake by plants, it was expected to result in higher N input than conventional methods for determination of N deposition (e.g. micrometeorological approaches, bulk N samplers). Using Lolium multiflorum and Eriophorum vaginatum as monitor plants and low, medium and high levels of fertilization, we aimed to simulate increasing N deposition to planted pots and to allocate airborne N after its uptake by the soil-plant system in aboveground biomass, roots and soil. Increasing N fertilization was positively correlated with biomass production of Eriophorum vaginatum, whereas atmospheric plant N uptake decreased and highest airborne N input of 899.8 ± 67.4 µg N d-1 pot-1 was found for low N fertilization. In contrast, Lolium multiflorum showed a clear dependency of N supply on plant N uptake and was highest (688.7 ± 41.4 µg N d-1 pot-1) for highly fertilized vegetation pots. Our results suggest that grass species respond differently to increasing N input. While crop grasses such as Lolium multiflorum take up N according to N availability, species adopted to nutrient-limited conditions like Eriophorum vaginatum show N saturation effects with increasing N supply. Total airborne N input ranged from about 24 to 66 kg N ha-1 yr-1 dependent on the used indicator plant and the amount of added fertilizer. Parallel determination of atmospheric N deposition using a micrometeorological approach

  6. Synthesis of 3H, 13C,2H3,15N and 14C-labelled SCH 466036, a histamine 3 receptor antagonist.

    PubMed

    Hesk, D; Borges, S; Dumpit, R; Hendershot, S; Koharski, D; Lavey, C; McNamara, P; Voronin, K

    2015-02-01

    The synthesis of [(3)H]SCH 466036, [Me-(3)H3]SCH 466036, [(13)C,(2)H3,(15)N]SCH 466036 and [(14)C]SCH 466036 is described. [(3)H]SCH 466036 was prepared in two steps via Raney Ni-catalysed exchange with tritiated water. [Me-(3)H3]SCH 466036 was prepared in a single step from [(3)H]methyl iodide in 45% yield. [(13)C,(2)H3,(15)N]SCH 466036 was prepared in two steps from [(15)N]hydroxylamine and [(13)C,(2)H3]methyl iodide with an overall yield of 16%. [(14)C]SCH 466036 was prepared in seven steps from [(14)C]potassium cyanide in an overall yield of 13%.

  7. Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.

    PubMed

    Hefke, Frederik; Bagaria, Anurag; Reckel, Sina; Ullrich, Sandra Johanna; Dötsch, Volker; Glaubitz, Clemens; Güntert, Peter

    2011-02-01

    We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273-6279 (1982)), types of amino acids are labeled with (13)C or/and (15)N such that cross peaks between (13)CO(i - 1) and (15)NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with (13)C and the second with (15)N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B(2)R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.

  8. Solid-state sup 13 C and sup 15 N NMR study of the low pH forms of bacteriorhodopsin

    SciTech Connect

    de Groot, H.J.; Smith, S.O.; Courtin, J.; van den Berg, E.; Winkel, C.; Lugtenburg, J.; Griffin, R.G.; Herzfeld, J. )

    1990-07-24

    The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved (epsilon-15N)lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the opsin shift. The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice.

  9. Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii*

    PubMed Central

    Mühlhaus, Timo; Weiss, Julia; Hemme, Dorothea; Sommer, Frederik; Schroda, Michael

    2011-01-01

    Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full 15N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform 15N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO2 and external acetate into protein biosynthesis, which also correlated with a rapid, but fully

  10. Fate of leaf-litter N in forest and grassland along a pedo-climatic gradient in south-western Siberia: an in situ 15N-labelling experiment

    NASA Astrophysics Data System (ADS)

    Brédoire, Félix; Zeller, Bernd; Nikitich, Polina; Barsukov, Pavel A.; Rusalimova, Olga; Bakker, Mark R.; Legout, Arnaud; Bashuk, Alexander; Kayler, Zachary E.; Derrien, Delphine

    2017-04-01

    The suitability of Siberia for agriculture is expected to increase in the next decades due to strong and rapid climatic changes, but little is known on the environmental drivers of soil fertility there, especially nitrogen (N). Plant-available N is mainly derived from litter decomposition. South-western (SW) Siberia is located on the transition between several bioclimatic zones that are predicted to shift and extend along with climate change (steppe, forest-steppe, sub-taiga). The soils of this region are formed on a common loess deposit but they are submitted to different climatic conditions and vegetation cover. In the south of the region, typically in steppe/forest-steppe, soil freezes over winter because of a relatively shallow snow-pack, and water shortages are frequent in summer. In the north, typically in sub-taiga, the soil is barely frozen in winter due to a thick snow-pack and sufficient soil moisture in summer. In this study, we characterized the dynamics of leaf litter decomposition and the transfer of N from leaf litter to the soil and back to plants. Four sites were chosen along a climate gradient (temperature, precipitation and snow depth). At each site, we applied 15N-labelled leaf litter on the soil surface in experimental plots in an aspen (Populus tremula L.) forest and in a grassland. Twice a year during three years, we tracked the 15N derived from the decomposing labelled-litter in the organic layers, in the first 15 cm of the soil, and in above-ground vegetation. Soil temperature and moisture were monitored at a daily timestep over three years and soil water budgets were simulated (BILJOU model, Granier et al. 1999). We observed contrasting patterns in the fate of litter-derived 15N between bioclimatic zones. Over three years, along with faster decay rates, the release of leaf litter-N was faster in sub-taiga than in forest-steppe. As such, higher quantities of 15N were transferred into the soil in sub-taiga. The transfer was also deeper there

  11. Alanine flux in obese and healthy humans as evaluated by /sup 15/N- and /sup 2/H/sub 3/-labeled alanines

    SciTech Connect

    Hoffer, L.J.; Yang, R.D.; Matthews, D.E.; Bistrian, B.R.; Bier, D.M.; Young, V.R.

    1988-10-01

    Estimates of plasma alanine flux as measured in humans using L-(/sup 15/N)-alanine or L-(3,3,3-/sup 2/H/sub 3/)alanine were compared by simultaneous intravenous infusion of both tracers. Plasma isotope enrichments were measured by chemical ionization gas chromatography-mass spectrometry. In 16 obese women before and during a hypocaloric diet and in 4 normal men in the postabsorptive and fed states, the fluxes were highly correlated (r2 = 0.93) although plasma alanine flux with the /sup 2/H tracer was two to three times greater than that obtained with (/sup 15/N)alanine. The fluxes decreased with the hypocaloric diet in obese subjects and increased during the fed state in healthy adults. Thus, although the estimates of alanine flux differed according to the tracer used, both appear to give equivalent information about changes in alanine kinetics induced by the nutritional conditions examined.

  12. Through-space (19) F-(15) N couplings for the assignment of stereochemistry in flubenzimine.

    PubMed

    Ghiviriga, Ion; Rubinski, Miles A; Dolbier, William R

    2016-07-01

    Through-space (19) F-(15) N couplings revealed the configuration of flubenzimine, with the CF3 group on N4 pointing towards the lone pair of N5. The (19) F-(15) N coupling constants were measured at natural abundance using a spin-state selective indirect-detection pulse sequence. As (15) N-labelled proteins are routinely synthesized for NMR studies, through-space (19) F-(15) N couplings have the potential to probe the stereochemistry of these proteins by (19) F labelling of some amino acids or can reveal the site of docking of fluorine-containing drugs. Copyright © 2016 John Wiley & Sons, Ltd.

  13. 15N Isotopic Crop Exchange Residue Studies Suggest that IPCC N Input Methodologies to Assess N2O-N Emissions Should be Reevaluated

    USDA-ARS?s Scientific Manuscript database

    It is difficult to quantify nitrogen (N) losses from agricultural systems to the environment, however we can use 15N isotopic techniques to conduct site specific studies to increase our knowledge about N management and fate. This manuscript synthesizes the review of two selected 15N isotopic studies...

  14. SIMS ion microscopy imaging of boronophenylalanine (BPA) and 13C15N-labeled phenylalanine in human glioblastoma cells: Relevance of subcellular scale observations to BPA-mediated boron neutron capture therapy of cancer

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash; Lorey, Daniel R., II

    2007-02-01

    p-Boronophenylalanine (BPA) is a clinically approved boron neutron capture therapy (BNCT) agent currently being used in clinical trials of glioblastoma multiforme, melanoma and liver metastases. Secondary ion mass spectrometry (SIMS) observations from the Cornell SIMS Laboratory provided support for using a 6 h infusion of BPA, instead of a 2 h infusion, for achieving higher levels of boron in brain tumor cells. These observations were clinically implemented in Phase II experimental trials of glioblastoma multiforme in Sweden. However, the mechanisms for higher BPA accumulation with longer infusions have remained unknown. In this work, by using 13C15N-labeled phenylalanine and T98G human glioblastoma cells, comparisons between the 10B-delivery of BPA and the accumulation of labeled phenylalanine after 2 and 6 h treatments were made with a Cameca IMS-3f SIMS ion microscope at 500 nm spatial resolution in fast frozen, freeze-fractured, freeze-dried cells. Due to the presence of the Na-K-ATPase in the plasma membrane of most mammalian cells, the cells maintain an approximately 10/1 ratio of K/Na in the intracellular milieu. Therefore, the quantitative imaging of these highly diffusible species in the identical cell in which the boron or labeled amino acid was imaged provides a rule-of-thumb criterion for validation of SIMS observations and the reliability of the cryogenic sampling. The labeled phenylalanine was detected at mass 28, as the 28(13C15N)- molecular ion. Correlative analysis with optical and confocal laser scanning microscopy revealed that fractured freeze-dried glioblastoma cells contained well-preserved ultrastructural details with three discernible subcellular regions: a nucleus or multiple nuclei, a mitochondria-rich perinuclear cytoplasmic region and the remaining cytoplasm. SIMS analysis revealed that the overall cellular signals of both 10B from BPA and 28CN- from labeled phenylalanine increased approximately 1.6-fold between the 2 and 6 h exposures

  15. Coupling Cover Crops with Alternative Swine Manure Application Strategies: Manure-15N Tracer Studies

    USDA-ARS?s Scientific Manuscript database

    Integration of rye cover crops with alternative liquid swine (Sus scrofa L.) manure application strategies may enhance retention of manure N in corn (Zea mays L.) - soybean [Glycine max (L.) Merr] cropping systems. The objective of this study was to quantify uptake of manure derived-N by a rye (Seca...

  16. 15N NMR study of nitrate ion structure and dynamics in hydrotalcite-like compounds

    USGS Publications Warehouse

    Hou, X.; James, Kirkpatrick R.; Yu, P.; Moore, D.; Kim, Y.

    2000-01-01

    We report here the first nuclear magnetic resonance (NMR) spectroscopic study of the dynamical and structural behavior of nitrate on the surface and in the interlayer of hydrotalcite-like compounds (15NO3--HT). Spectroscopically resolvable surface-absorbed and interlayer NO3- have dramatically different dynamical characteristics. The interlayer nitrate shows a well defined, temperature independent uniaxial chemical shift anisotropy (CS A) powder pattern. It is rigidly held or perhaps undergoes rotation about its threefold axis at all temperatures between -100 ??C and +80 ??C and relative humidities (R.H.) from 0 to 100% at room temperature. For surface nitrate, however, the dynamical behavior depends substantially on temperature and relative humidity. Analysis of the temperature and R.H. dependences of the peak width yields reorieritational frequencies which increase from essentially 0 at -100 ??C to 2.6 ?? 105 Hz at 60 ??C and an activation energy of 12.6 kJ/mol. For example, for samples at R.H. = 33%, the surface nitrate is isotropically mobile at frequencies greater than 105 Hz at room temperature, but it becomes rigid or only rotates on its threefold axis at -100 ??C. For dry samples and samples heated at 200 ??C (R.H. near 0%), the surface nitrate is not isotropically averaged at room temperature. In contrast to our previous results for 35Cl--containing hydrotalcite (35Cl--HT), no NMR detectable structural phase transition is observed for 15NO3--HT. The mobility of interlayer nitrate in HT is intermediate between that of carbonate and chloride.

  17. Salt marsh ecosystem biogeochemical responses to nutrient enrichment: a paired 15N tracer study.

    PubMed

    Drake, D C; Peterson, Bruce J; Galván, Kari A; Deegan, Linda A; Hopkinson, Charles; Johnson, J Michael; Koop-Jakobsen, K; Lemay, Lynsey E; Picard, Christian

    2009-09-01

    We compared processing and fate of dissolved NO3- in two New England salt marsh ecosystems, one receiving natural flood tide concentrations of approximately 1-4 micromol NO3-/ L and the other receiving experimentally fertilized flood tides containing approximately 70-100 micromol NO3-/ L. We conducted simultaneous 15NO3- (isotope) tracer additions from 23 to 28 July 2005 in the reference (8.4 ha) and fertilized (12.4 ha) systems to compare N dynamics and fate. Two full tidal cycles were intensively studied during the paired tracer additions. Resulting mass balances showed that essentially 100% (0.48-0.61 mol NO3-N.ha(-1).h(-1)) of incoming NO3- was assimilated, dissimilated, sorbed, or sedimented (processed) within a few hours in the reference system when NO3- concentrations were 1.3-1.8 micromol/L. In contrast, only 50-60% of incoming NO3- was processed in the fertilized system when NO3- concentrations were 84-96 micromol/L; the remainder was exported in ebb tidewater. Gross NO3- processing was approximately 40 times higher in the fertilized system at 19.34-24.67 mol NO3-N.ha(-1).h(-1). Dissimilatory nitrate reduction to ammonium was evident in both systems during the first 48 h of the tracer additions but <1% of incoming 15NO3- was exported as 15NH4+. Nitrification rates calculated by 15NO3- dilution were 6.05 and 4.46 mol.ha(-1).h(-1) in the fertilized system but could not be accurately calculated in the reference system due to rapid (<4 h) NO3- turnover. Over the five-day paired tracer addition, sediments sequestered a small fraction of incoming NO3-, although the efficiency of sequestration was 3.8% in the reference system and 0.7% in the fertilized system. Gross sediment N sequestration rates were similar at 13.5 and 12.6 mol.ha(-1).d(-1), respectively. Macrophyte NO3- uptake efficiency, based on tracer incorporation in aboveground tissues, was considerably higher in the reference system (16.8%) than the fertilized system (2.6%), although bulk uptake of NO3

  18. The fates of (15)N-labeled fertilizer in a wheat-soil system as influenced by fertilization practice in a loamy soil.

    PubMed

    Chen, Zhaoming; Wang, Huoyan; Liu, Xiaowei; Lu, Dianjun; Zhou, Jianmin

    2016-10-07

    Appropriate fertilization practice is crucial to achieve maximum wheat grain yield with minimum nitrogen (N) loss. A field (15)N micro-plot experiment was conducted to determine the effects of application methods [split application (SA) and band application (BA)] and N rates (60, 150 and 240 kg ha(-1)) on the wheat grain yield, urea-(15)N fate and N efficiency in Jiangyan County, China. At high N rates, wheat grain yield was significantly higher for SA than BA treatment, but there was no difference at the lower N rates. Plant N derived from fertilizer was higher in SA than in BA treatment. The high N fertilizer application increased total N uptake by wheat derived from fertilizer, but wheat plant N derived from soil was not affected by the N rate. Fertilizer-N recovery in SA treatment was higher than in BA treatment. Residual N recovery in the 0-80 cm soil layer was 31-51%, which decreased with increasing N rate. The highest N loss was found for BA treatment at the N application of 240 kg ha(-1). The one-time BA of N fertilizer, especially for higher N rates, led to reduced wheat grain yield and N efficiency, and increased the N loss.

  19. The fates of 15N-labeled fertilizer in a wheat–soil system as influenced by fertilization practice in a loamy soil

    PubMed Central

    Chen, Zhaoming; Wang, Huoyan; Liu, Xiaowei; Lu, Dianjun; Zhou, Jianmin

    2016-01-01

    Appropriate fertilization practice is crucial to achieve maximum wheat grain yield with minimum nitrogen (N) loss. A field 15N micro-plot experiment was conducted to determine the effects of application methods [split application (SA) and band application (BA)] and N rates (60, 150 and 240 kg ha−1) on the wheat grain yield, urea-15N fate and N efficiency in Jiangyan County, China. At high N rates, wheat grain yield was significantly higher for SA than BA treatment, but there was no difference at the lower N rates. Plant N derived from fertilizer was higher in SA than in BA treatment. The high N fertilizer application increased total N uptake by wheat derived from fertilizer, but wheat plant N derived from soil was not affected by the N rate. Fertilizer-N recovery in SA treatment was higher than in BA treatment. Residual N recovery in the 0–80 cm soil layer was 31–51%, which decreased with increasing N rate. The highest N loss was found for BA treatment at the N application of 240 kg ha−1. The one-time BA of N fertilizer, especially for higher N rates, led to reduced wheat grain yield and N efficiency, and increased the N loss. PMID:27713476

  20. The fates of 15N-labeled fertilizer in a wheat–soil system as influenced by fertilization practice in a loamy soil

    NASA Astrophysics Data System (ADS)

    Chen, Zhaoming; Wang, Huoyan; Liu, Xiaowei; Lu, Dianjun; Zhou, Jianmin

    2016-10-01

    Appropriate fertilization practice is crucial to achieve maximum wheat grain yield with minimum nitrogen (N) loss. A field 15N micro-plot experiment was conducted to determine the effects of application methods [split application (SA) and band application (BA)] and N rates (60, 150 and 240 kg ha‑1) on the wheat grain yield, urea-15N fate and N efficiency in Jiangyan County, China. At high N rates, wheat grain yield was significantly higher for SA than BA treatment, but there was no difference at the lower N rates. Plant N derived from fertilizer was higher in SA than in BA treatment. The high N fertilizer application increased total N uptake by wheat derived from fertilizer, but wheat plant N derived from soil was not affected by the N rate. Fertilizer-N recovery in SA treatment was higher than in BA treatment. Residual N recovery in the 0–80 cm soil layer was 31–51%, which decreased with increasing N rate. The highest N loss was found for BA treatment at the N application of 240 kg ha‑1. The one-time BA of N fertilizer, especially for higher N rates, led to reduced wheat grain yield and N efficiency, and increased the N loss.

  1. Dynamics of Nitrogen Uptake and Mobilization in Field-grown Winter Oilseed Rape (Brassica napus) From Stem Extension to Harvest. II. An 15N-labelling-based Simulation Model of N Partitioning Between Vegetative and Reproductive Tissues

    PubMed Central

    MALAGOLI, P.; LAINE, P.; ROSSATO, L.; OURRY, A.

    2005-01-01

    • Background and Aims Oilseed rape (Brassica napus) has often been used as a catch crop to deal with the issue of N leaching, but for this to be effective, prediction of the crop's N uptake capability and N partitioning is required. The aim of this work was to build a compartmental model of N dynamics in oilseed rape, based on the kinetic description of N uptake, partitioning and mobilization in each organ. • Model In this study, logistic and exponential equations were fitted to the N relations of each compartment, especially the leaf at each node. Data previously obtained from an 15N-labelling field experiment was used to quantify the partitioning of total N content, the allocation of N taken up and subsequent changes in the sink/source status for endogenous N in each tissue throughout the growth cycle. • Key Results and Conclusions This modelling approach provides a unique tool for the quantitative estimation of cycling of endogenous N in relation to changes in N uptake at the whole-plant level. Furthermore, as oilseed rape is known to release large amounts of N to the soil during spring through leaf loss, this model was used to identify potential methods for improving the N harvest index of the crop. Simulations showed that N content or yield could be improved by 15 % by optimizing N transfer from vegetative to reproductive tissues and by reducing the residual %N (DW) in abscised leaves. PMID:15802311

  2. Isotope labeling for NMR studies of macromolecular structure and interactions

    SciTech Connect

    Wright, P.E.

    1994-12-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.

  3. Structure and reactivity of lithium amides. /sup 6/Li, /sup 13/C, and /sup 15/N NMR spectroscopic studies and colligative measurements of lithium diphenylamide and lithium diphenylamide-lithium bromide complex solvated by tetrahydrofuran

    SciTech Connect

    DePue, J.S.; Collum, D.B.

    1988-08-03

    /sup 6/Li, /sup 13/C, and /sup 15/N NMR spectroscopic studies of lithium diphenylamide in THF/hydrocarbon solutions (THF = tetrahydrofuran) detected two different species. /sup 6/Li and /sup 15/N NMR spectroscopic studies of (/sup 6/Li, /sup 15/N)lithium diphenylamide showed the species observed at low THF concentrations to be a cyclic oligomer. Structural analogies provided strong support for a dimer while colligative measurements at 0/degrees/C indicated the dimer to be di- or trisolvated. On the basis of the observed mass action effects, the species appearing at intermediate THF concentrations is assigned as a contact or solvent-separated ion-paired monomer. Lithium diphenylamide forms a 1:1 adduct with lithium bromide at low THF concentrations. A combination of /sup 6/Li-/sup 15/N double labeling studies and colligative measurements supports a trisolvated cyclic mixed dimer structure. Although detailed spectroscopic studies at elevated THF concentrations were precluded by high fluctionality, the similarity of the /sup 13/C chemical shifts of lithium diphenylamide in the presence and absence of lithium bromide provide indirect evidence that the mixed dimer undergoes a THF concentration dependent dissociation to the monomeric amide and free lithium bromide. 24 references, 9 figures, 2 tables.

  4. Pathways of nitrogen assimilation in cowpea nodules studied using /sup 15/N/sub 2/ and allopurinol. [Vigna unguiculata L. Walp. cv Vita

    SciTech Connect

    Atkins, C.A.; Storer, P.J.; Pate, J.S.

    1988-01-01

    In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo (3,4-d)pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (vigna unguiculata L. Walp. cv Vita 3) formed (/sup 15/N)xanthine from /sup 15/N/sub 2/ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.87.99.2) activity. Negligible /sup 15/N -labeling of asparagine from /sup 15/N/sub 2/ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.

  5. Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.

    PubMed

    Landrieu, I; Casteels, P; Odaert, B; De Veylder, L; Portetelle, D; Lippens, G; Van Montagu, M; Inzé, D

    1999-06-01

    The CKS1At gene product, p10CKS1At from Arabidopsis thaliana, is a member of the cyclin-dependent kinase subunit (CKS) family of small proteins. These proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and play an essential, but still not precisely known role in cell cycle progression. To solve the structure of p10CKS1At, a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) spectroscopy. The first attempt to express CKS1At in Escherichia coli under the control of the T7 promoter was not successful. E. coli BL21(DE3) cotransformed with the CKS1At gene and the E. coli argU gene that encoded the arginine acceptor tRNAUCU produced a sufficient amount of p10CKS1At to start the structural study by NMR. Replacement of four rare codons in the CKS1At gene sequence, including a tandem arginine, by highly used codons in E. coli, restored also a high expression of the recombinant protein. Double-isotopic enrichment by 13C and 15N is reported that will facilitate the NMR study. Isotopically labeled p10CKS1At was purified to yield as much as 55 mg from 1 liter of minimal media by a two-step chromatographic procedure. Preliminary results of NMR spectroscopy demonstrate that a full structural analysis using triple-resonance spectra is feasible for the labeled p10CKS1At protein. Copyright 1999 Academic Press.

  6. Fate of nitrogen in riparian forest soils and trees: an 15N tracer study simulating salmon decay.

    PubMed

    Drake, Deanne C; Naiman, I Robert J; Bechtold, J Scott

    2006-05-01

    We introduced an 15N-NH4+ tracer to the riparian forest of a salmon-bearing stream (Kennedy Creek, Washington, USA) to quantify the cycling and fate of a late-season pulse of salmon N and, ultimately, mechanisms regulating potential links between salmon abundance and tree growth. The 15N tracer simulated deposition of 7.25 kg of salmon (fresh) to four 50-m2 plots. We added NH4+ (the initial product of salmon carcass decay) and other important nutrients provided by carcasses (P, S, K, Mg, Ca) to soils in late October 2003, coincident with local salmon spawning. We followed the 15N tracer through soil and tree pools for one year. Biological uptake of the 15N tracer occurred quickly: 64% of the 15N tracer was bound in soil microbiota within 14 days, and roots of the dominant riparian tree, western red cedar (Thuja plicata), began to take up 15N tracer within seven days. Root uptake continued through the winter. The 15N tracer content of soil organic matter reached a maximum of approximately 52%, five weeks after the application, and a relative equilibrium of approximately 40% within five months. Six months after the addition, in spring 2004, at least 37% of the 15N tracer was found in tree tissues: approximately 23% in foliage, approximately 11% in roots, and approximately 3% in stems. Within the stems, xylem and phloem sap contained approximately 96% of the tracer N, and approximately 4% was in structural xylem N. After one year, at least 28% of the 15N tracer was still found in trees, and loss from the plots was only approximately 20%. The large portion of tracer N taken up in the fall and reallocated to leaves and stems the following spring provides mechanistic evidence for a one-year-lagged tree-growth response to salmon nutrients. Salmon nutrients have been deposited in the Kennedy Creek system each fall for centuries, but the system shows no evidence of nutrient saturation. Rates of N uptake and retention are a function of site history and disturbance and also

  7. δ 15N Studies of Nitrogen Use by the Red Mangrove, Rhizophora mangle L. in South Florida

    NASA Astrophysics Data System (ADS)

    Fry, B.; Bern, A. L.; Ross, M. S.; Meeder, J. F.

    2000-02-01

    To help define nitrogen (N) sources and patterns of N processing in mangrove ecosystems, mangrove leaf nitrogen contents and δ 15N values were assayed in three marshes along the south Florida coast. In each marsh, leaf samples were collected from dwarf mangroves at interior locations and taller mangroves at the ocean fringe. Leaf % N and δ 15N values did not differ consistently between dwarf and tall mangroves, even though there were large variations in δ 15N (18‰ range, -5 to +13‰) and % N (1·2% range, 0·9-2·1%). Highest % N and δ 15N values occurred along the western margin of Biscayne Bay where canals draining agricultural lands deliver high-nitrate waters to fringing mangrove marshes. High mangrove δ 15N values may be good biomonitors of anthropogenic N loading to south Florida estuaries. Lower values likely reflect less anthropogenic N entering the mangrove marshes, as well as differences in plant physiology that occur along the fringe-dwarf gradient.

  8. Synthesis of 15N omega-hydroxy-L-arginine and ESR and 15N-NMR studies for the elucidation of the molecular mechanism of enzymic nitric oxide formation from L-arginine.

    PubMed

    Clement, B; Schnörwangen, E; Kämpchen, T; Mordvintcev, P; Mülsch, A

    1994-12-01

    N omega-Hydroxy-L-arginine (2) was prepared by a multi-stage synthesis; the key step was the addition of hydroxylamine to the protected cyanamide 8. The presence of N-hydroxyguanidines was confirmed, above all, by 15N-NMR investigations. 15N omega-Hydroxy-L-arginine (2) was converted quantitatively to 15NO by NO synthases from macrophages. 15NO was identified by ESR-spectroscopy. These experiments confirm that 15N omega-hydroxy-L-arginine (2) is an intermediate in the biosynthesis of NO from arginine (1) and that the N-hydroxylated N-atom is present in the NO formed.

  9. Are vascular epiphytes nitrogen or phosphorus limited? A study of plant (15) N fractionation and foliar N : P stoichiometry with the tank bromeliad Vriesea sanguinolenta.

    PubMed

    Wanek, Wolfgang; Zotz, Gerhard

    2011-10-01

    Although there is unambiguous evidence for vascular epiphytic plants to be limited by insufficient water and nutrient supply under natural conditions, it is an open debate whether they are primarily phosphorus (P) or nitrogen (N) limited. Plant (15) N fractionation and foliar N : P stoichiometry of a tank epiphyte (Vriesea sanguinolenta), and its response to combined N-P fertilization, were studied under semi-natural conditions over 334 d to clarify the type of nutrient limitation. Plants collected in the field and experimental plants with limited nutrient supply showed significant plant (15) N fractionation (mean 5‰) and plant N : P ratios of c. 13.5. Higher relative growth rates and declines in plant (15) N fractionation (0.5‰) and in foliar N : P ratios to 8.5 in the high N-P treatment indicated that these epiphytes were P limited in situ. The critical foliar N : P ratio was 10.4, as derived from the breakpoint in the relationship between plant (15) N fractionation and foliar N : P. We interpret the widespread (15) N depletion of vascular epiphytes relative to their host trees as deriving from (15) N fractionation of epiphytes as a result of P limitation. High foliar N : P ratios (> 12) corroborate widespread P limitation (or co-limitation by N and P) of epiphytic bromeliads and, possibly, other epiphyte species.

  10. [Studies on the effect of phosphoric phenyl ester diamide as inhibitor of the rumen urease of dairy cows. 2. The metabolism of 15N-urea].

    PubMed

    Voigt, J; Krawielitzki, K; Piatkowski, B

    1980-12-01

    Two lactating dairy cows supplied with rumen and duodenal re-entrance cannulae received with their rations containing 8.2% vegetable crude protein 180 g urea per day (I) or urea treated with the urease inhibitor phosphoric phenyl ester diamide (PPD, 1% of the N-quota). The cows, accustomed to urea, received the PPD-urea without adaptation (II) and after a 30-day adaptation (III) to PPD. On the first day of the experiment one half of the urea was given ruminally in its 15N-labelled form. 2 h after the isotope supplementation 62.5 and 24 mg NH3 and 6.58 and 21 mg urea/100 ml could be detected in the rumen juice of I to III. Within 72 h 16.6, 26.1 and 25.2% of the 15N-excess given (15N') passed the duodenum in TCA - soluble form and 31.2, 28.4 and 41.7% in TCA - precipitable form. 15.6, 24.4 and 21.5% of the total amount of 15N were excreted in urine and 4.5, 4.6 and 6.0% in the milk protein. The values for faeces were 14.4, 14,4 and 15.4%. The conclusion from these results and from the dynamics of the relative 15N' in the fractions of the rumen fluid is that with a limited inhibition of rumen urease by PPD as it develops after the adaptation, the utilisation of urea-N can be improved.

  11. sup 15 N and sup 13 C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

    SciTech Connect

    Jaffe, E.K.; Rajagopalan, J.S. ); Markham, G.D. )

    1990-09-11

    Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through {sup 13}C and {sup 15}N NMR. The authors knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by {sup 13}C or {sup 15}N NMR. Here they extend their {sup 13}C NMR studies to PBGS complexes with (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA and report {sup 15}N NMR studies of ({sup 15}N)ALA bound to PBGS. As in their previous {sup 13}C NMR studies, observation of enzyme-bound {sup 15}N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pK{sub a} is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent. For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C{sub 4} of ALA and an active-site lysine. The {sup 13}C chemical shift of (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between ({sup 15}N)ALA and hydrazine or hydroxylamine, the {sup 15}N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C{sub 4} of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.

  12. Effect of protein restriction on (15)N transfer from dietary [(15)N]alanine and [(15)N]Spirulina platensis into urea.

    PubMed

    Hamadeh, M J; Hoffer, L J

    2001-08-01

    Six normal men consumed a mixed test meal while adapted to high (1.5 g. kg(-1) x day(-1)) and low (0.3 g. kg(-1) x day(-1)) protein intakes. They completed this protocol twice: when the test meals included 3 mg/kg of [(15)N]alanine ([(15)N]Ala) and when they included 30 mg/kg of intrinsically labeled [(15)N]Spirulina platensis ([(15)N]SPI). Six subjects with insulin-dependent diabetes mellitus (IDDM) receiving conventional insulin therapy consumed the test meal with added [(15)N]Ala while adapted to their customary high-protein diet. Protein restriction increased serum alanine, glycine, glutamine, and methionine concentrations and reduced those of leucine. Whether the previous diet was high or low in protein, there was a similar increase in serum alanine, methionine, and branched-chain amino acid concentrations after the test meal and a similar pattern of (15)N enrichment in serum amino acids for a given tracer. When [(15)N]Ala was included in the test meal, (15)N appeared rapidly in serum alanine and glutamine, to a minor degree in leucine and isoleucine, and not at all in other circulating amino acids. With [(15)N]SPI, there was a slow appearance of the label in all serum amino acids analyzed. Despite the different serum amino acid labeling, protein restriction reduced the postmeal transfer of dietary (15)N in [(15)N]Ala or [(15)N]SPI into [(15)N]urea by similar amounts (38 and 43%, respectively, not significant). The response of the subjects with IDDM was similar to that of the normal subjects. Information about adaptive reductions in dietary amino acid catabolism obtained by adding [(15)N]Ala to a test meal appears to be equivalent to that obtained using an intrinsically labeled protein tracer.

  13. 15N nuclear magnetic resonance studies of acid-base properties of pyridoxal-5'-phosphate aldimines in aqueous solution.

    PubMed

    Sharif, Shasad; Huot, Monique Chan; Tolstoy, Peter M; Toney, Michael D; Jonsson, K Hanna M; Limbach, Hans-Heinrich

    2007-04-19

    By use of 15N NMR spectroscopy, we have measured the pKa values of the aldimines 15N-(pyridoxyl-5'-phosphate-idine)-methylamine (2a), N-(pyridoxyl-5'-phosphate-15N-idine)-methylamine (2b), and 15N-(pyridoxyl-idine)-methylamine (3). These aldimines model the cofactor pyridoxal-5'-phosphate (PLP, 1) in a variety of PLP-dependent enzymes. The acid-base properties of the aldimines differ substantially from those of the free cofactor in the aldehyde form 1a or in the hydrated form 1b, which were also investigated using 15N NMR for comparison. All compounds contain three protonation sites, the pyridine ring, the phenol group, and the side chain phosphate (1, 2) or hydroxyl group (3). In agreement with the literature, 1a exhibits one of several pKas at 2.9 and 1b at 4.2. The 15N chemical shifts indicate that the corresponding deprotonation occurs partially in the pyridine and partially in the phenolic site, which compete for the remaining proton. The equilibrium constant of this ring-phenolate tautomerism was measured to be 0.40 for 1a and 0.06 for 1b. The tautomerism is essentially unaltered above pH 6.1, where the phosphate group is deprotonated to the dianion. This means that the pyridine ring is more basic than the phenolate group. Pyridine nitrogen deprotonation occurs at 8.2 for 1a and at 8.7 for 1b. By contrast, above pH 4 the phosphate site of 2 is deprotonated, while the pyridine ring pKa is 5.8. The Schiff base nitrogen does not deprotonate below pH 11.4. When the phosphate group is removed, the pKa of the Schiff base nitrogen decreases to 10.5. The phenol site cannot compete for the proton of the Schiff base nitrogen and is present in the entire pH range as a phenolate, preferentially hydrogen bonded to the solvent. The intrinsic 15N chemical shifts provide information about the hydrogen bond structures of the protonated and unprotonated species involved. Evidence is presented that the intramolecular OHN hydrogen bond of PLP aldimines is broken in aqueous

  14. Nitrogen assimilation and short term retention in a nutrient-rich tidal freshwater marsh - a whole ecosystem 15N enrichment study

    NASA Astrophysics Data System (ADS)

    Gribsholt, B.; Struyf, E.; Tramper, A.; de Brabandere, L.; Brion, N.; van Damme, S.; Meire, P.; Dehairs, F.; Middelburg, J. J.; Boschker, H. T. S.

    2007-01-01

    An intact tidal freshwater marsh system (3477 m2) was labelled by adding 15N-ammonium as a tracer to the flood water inundating the ecosystem. The appearance and retention of 15N-label in different marsh components (leaves, roots, sediment, leaf litter and invertebrate fauna) was followed over 15 days. This allowed us to elucidate the direct assimilation and dependence on creek-water nitrogen on a relatively short term and provided an unbiased assessment of the relative importance of the various compartments within the ecosystem. Two separate experiments were conducted, one in spring/early summer (May 2002) when plants were young and building up biomass; the other in late summer (September 2003) when macrophytes were in a flowering or early senescent state. Nitrogen assimilation rate (per hour inundated) was >3 times faster in May compared to September. On both occasions, however, the results clearly revealed that the less conspicuous compartments such as leaf litter and ruderal vegetations are more important in nitrogen uptake and retention than the prominent reed (Phragmites australis) meadows. Moreover, short-term nitrogen retention in these nutrient rich marshes occurs mainly via microbial pathways associated with the litter and sediment. Rather than direct uptake by macrophytes, it is the large reactive surface area provided by the tidal freshwater marsh vegetation that is most crucial for nitrogen transformation, assimilation and short term retention in nutrient rich tidal freshwater marshes. Our results clearly revealed the dominant role of microbes in initial nitrogen retention in marsh ecosystems.

  15. Kinetic 15N-isotope effects on algal growth

    NASA Astrophysics Data System (ADS)

    Andriukonis, Eivydas; Gorokhova, Elena

    2017-03-01

    Stable isotope labeling is a standard technique for tracing material transfer in molecular, ecological and biogeochemical studies. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism metabolism and growth, which is not consistent with current theoretical and empirical knowledge on kinetic isotope effects. Here, we demonstrate profound changes in growth dynamics of the green alga Raphidocelis subcapitata grown in 15N-enriched media. With increasing 15N concentration (0.37 to 50 at%), the lag phase increased, whereas maximal growth rate and total yield decreased; moreover, there was a negative relationship between the growth and the lag phase across the treatments. The latter suggests that a trade-off between growth rate and the ability to adapt to the high 15N environment may exist. Remarkably, the lag-phase response at 3.5 at% 15N was the shortest and deviated from the overall trend, thus providing partial support to the recently proposed Isotopic Resonance hypothesis, which predicts that certain isotopic composition is particularly favorable for living organisms. These findings confirm the occurrence of KIE in isotopically enriched algae and underline the importance of considering these effects when using stable isotope labeling in field and experimental studies.

  16. Kinetic 15N-isotope effects on algal growth

    PubMed Central

    Andriukonis, Eivydas; Gorokhova, Elena

    2017-01-01

    Stable isotope labeling is a standard technique for tracing material transfer in molecular, ecological and biogeochemical studies. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism metabolism and growth, which is not consistent with current theoretical and empirical knowledge on kinetic isotope effects. Here, we demonstrate profound changes in growth dynamics of the green alga Raphidocelis subcapitata grown in 15N-enriched media. With increasing 15N concentration (0.37 to 50 at%), the lag phase increased, whereas maximal growth rate and total yield decreased; moreover, there was a negative relationship between the growth and the lag phase across the treatments. The latter suggests that a trade-off between growth rate and the ability to adapt to the high 15N environment may exist. Remarkably, the lag-phase response at 3.5 at% 15N was the shortest and deviated from the overall trend, thus providing partial support to the recently proposed Isotopic Resonance hypothesis, which predicts that certain isotopic composition is particularly favorable for living organisms. These findings confirm the occurrence of KIE in isotopically enriched algae and underline the importance of considering these effects when using stable isotope labeling in field and experimental studies. PMID:28281640

  17. A 1H, 13C and 15N NMR study in solution and in the solid state of six N-substituted pyrazoles and indazoles.

    PubMed

    Claramunt, Rosa M; Santa María, M Dolores; Sanz, Dionisia; Alkorta, Ibon; Elguero, José

    2006-05-01

    Three N-substituted pyrazoles and three N-substituted indazoles [1-(4-nitrophenyl)-3,5-dimethylpyrazole (1), 1-(2,4-dinitrophenyl)-3,5-dimethylpyrazole (2), 1-tosyl-pyrazole (3), 1-p-chlorobenzoylindazole (4), 1-tosylinda-zole (5) and 2-(2-hydroxy-2-phenylethyl)-indazole (6)] have been studied by NMR spectroscopy in solution (1H, 13C, 15N) and in the solid state (13C, 15N). The chemical shifts have been compared with GIAO/DFT calculated absolute shieldings. Some discrepancies have been analyzed.

  18. Nitrate degradation without 15N enrichment: a hydrochemical and isotopic study of a fractured rock aquifer including embedded lakes.

    PubMed

    Bozau, Elke; Knöller, Kay; Strauch, Gerhard

    2006-09-01

    Water samples from three quarry lakes and the surrounding fractured rock aquifer were investigated for delta18O and delta2H (H2O), delta15N and delta18O (NO3-), as well as anions and cations. Lake water and groundwater can be distinguished by their different chemical and isotopic composition. Because of evaporation processes, 18O and 2H are enriched in the lake water and can be used as natural tracers for the water dynamic of the lakes. The groundwater is characterised by high nitrate concentrations (up to 120 mg/l). Lake internal processes reduce the nitrate concentration in the quarry lakes. However, no enrichment of delta15N and delta18O in nitrate, typical for microbial nitrate degradation, is observed in the lake water. Because of the complex flow paths in the fractured rock aquifer and the intense chemical transformations at the interface between groundwater and lake water, isotopic and hydrochemical data of lake water and groundwater alone do not conclusively explain hydrological and hydrochemical processes of the investigated lake-groundwater system.

  19. 15N solid-state nuclear magnetic resonance study of pyrolyzed metal-polyaniline cathode catalysts for oxygen reduction in fuel cells

    NASA Astrophysics Data System (ADS)

    Kuroki, Shigeki; Hosaka, Yo; Yamauchi, Chiharu; Nagata, Shinsuke; Sonoda, Mayu

    2015-09-01

    The oxygen reduction reaction (ORR) activity of pyrolyzed metal-free and metal (Mn, Fe, Co, Ni and Cu)-containing polyaniline (PANI) in polymer electrolyte fuel cell (PEFC) was studied. The metal-free PANI800 shows quite poor ORR catalytic activity, whilst the metal-containing PANIMe800 display a better ORR activity. The 15N CP/MAS NMR spectra of PANINi800 and PANICu800 show one weak peak at 118 ppm and there is no peak observed in PANIFe800, against that of PANI800, PANIMn800, PANICo800 and PANINi800 show two peaks at 273 and 118 ppm assigned to the pyridinic and pyridinium nitrogens. It is because of the paramagnetic effect of metal ions. The 15N spin-echo NMR spectra of PANIMe800 with fast recycle delay show the peaks at 140 and 270 ppm assigned to the graphitic and pyridinic nitrogens, against that of PANI800 shows no peak. The spectra of PANIMn800, PANICo800, PANINi800 and PANICu600 also contain a very broaden peak at 430 ppm assigned to the nitrogen with Fermi-contact effect from metal ions. The spectra of PANIFe800 show some spinning side bands and the average Fe3+-15N distance can be calculated. The some amount of iron ion are relieved and average Fe3+-15N distance increase after acid washing and the ORR activity decreases.

  20. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons.

    PubMed

    Live, D H; Cowburn, D; Breslow, E

    1987-10-06

    NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons

    SciTech Connect

    Live, D.H.; Cowburn, D.

    1987-10-06

    NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, /sup 15/N labeling being used to identify specific backbone /sup 15/N and /sup 1/H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence for hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neutrophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of /sup 15/N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone. The results suggest significant conformational alteration in neurophysin-hormone complexes at low pH possibly associated with protonation of the carboxyl group of the hormone-protein salt bridge.

  2. Relationship between systemic drug absorption and gastrointestinal transit after the simultaneous oral administration of carbamazepine as a controlled-release system and as a suspension of 15N-labelled drug to healthy volunteers.

    PubMed

    Wilding, I R; Davis, S S; Hardy, J G; Robertson, C S; John, V A; Powell, M L; Leal, M; Lloyd, P; Walker, S M

    1991-11-01

    1. Plasma drug concentrations after a single oral administration of a suspension of carbamazepine (CBZ) and a 20/200 CBZ Oros osmotic pump system were measured in eight healthy male volunteers. The oral suspension contained 100 mg CBZ labelled with the stable isotope nitrogen-15, whilst the Oros contained 200 mg unlabelled CBZ. Plasma concentrations of [15N]-CBZ and CBZ were measured simultaneously by gas chromatography-mass spectrometry. 2. The position of the CBZ Oros (labelled with indium-111) in the gastrointestinal tract was followed by gamma scintigraphy. Plasma drug concentrations after the two treatments were used to relate pharmacokinetic with transit data. 3. The Oros was taken after breakfast and gastric emptying occurred between 1.1- greater than h post-dosing (median, 5.3 h). Small intestinal transit times ranged from 1.5- greater than 3.6 h, with a median of 2.2 h. There were wide individual variations in colonic transit, and the total transit time ranged from 10-60 h (median, 22 h). 4. Relative systemic bioavailability of CBZ from the Oros was reduced compared with that from the suspension (mean dose normalised AUC ratio = 0.69 +/- 0.17; mean dose-normalised AUC ratio = 0.85 +/- 0.13, allowing for actual release from the Oros system). 5. The in vivo absorption of drug into the systemic circulation from the Oros was estimated using the Wagner-Nelson method. This showed that absorption of CBZ was rapid when the Oros was present in the stomach and small intestine, the rate being determined by the release of drug from the system.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Preparation and characterization of 15N-enriched, size-defined heparan sulfate precursor oligosaccharides

    PubMed Central

    Sigulinsky, Crystal; Babu, Ponnusamy; Victor, Xylophone V.; Kuberan, Balagurunathan

    2009-01-01

    We report the preparation of size-defined [15N]N-acetylheparosan oligosaccharides from Escherichia coli-derived 15N-enriched N-acetylheparosan. Optimized growth conditions of E. coli in minimal media containing 15NH4Cl yielded [15N]N-acetylheparosan on a preparative scale. Depolymerization of [15N]N-acetylheparosan by heparitinase I yielded resolvable, even-numbered oligosaccharides ranging from disaccharide to icosaccharide. Anion-exchange chromatography-assisted fractionation afforded size-defined [15N]N-acetylheparosan oligosaccharides identifiable by ESI-TOFMS. These isotopically labeled oligosaccharides will prove to be valuable research tools for the chemoenzymatic synthesis of heparin and heparan sulfate oligosaccharides and for the study of their structural biology. PMID:19945695

  4. A study on the influence of fast amide exchange on the accuracy of (15)N relaxation rate constants.

    PubMed

    Jurt, Simon; Zerbe, Oliver

    2012-12-01

    (15)N relaxation rates of amide moieties provide insight both into global as well as local backbone dynamics of peptides and proteins. As the differences in the relaxation rates in general are small, their accurate determination is of prime importance. One potential source of error is fast amide exchange. It is well known that in its presence the effects of saturation transfer and H/D exchange may result in erroneous apparent relaxation rates R (1) and R (2). Here, the extent of these errors is rigorously examined. Theoretical considerations reveal that even when saturation effects are absent, H/D exchange will easily result in significant deviations from the true values. In particular overestimations of up to 10 % in R (1) and up to 5 % in R (2) are observed. An alternative scheme for fitting the relaxation data to the corresponding exponentials is presented that in the best cases not only delivers more accurate relaxation rates but also allows extracting estimates for the exchange rates. The theoretical computations were tested and verified for the case of ubiquitin.

  5. Backbone dynamics of (1-71)- and (1-36)bacterioopsin studied by two-dimensional (1)H- (15)N NMR spectroscopy.

    PubMed

    Orekhov, V Y; Pervushin, K V; Korzhnev, D M; Arseniev, A S

    1995-09-01

    The backbone dynamics of uniformly (15)N-labelled fragments (residues 1-71 and 1-36) of bacterioopsin, solubilized in two media (methanol-chloroform (1:1), 0.1 M (2)HCO(2)NH(4), or SDS micelles) have been investigated using 2D proton-detected heteronuclear (1)H-(15)N NMR spectroscopy at two spectrometer frequencies, 600 and 400 MHz. Contributions of the conformational exchange to the transverse relaxation rates of individual nitrogens were elucidated using a set of different rates of the CPMG spin-lock pulse train and were essentially suppressed by the high-frequency CPMG spin-lock. We found that most of the backbone amide groups of (1-71)bacterioopsin in SDS micelles are involved in the conformational exchange process over a rate range of 10(3) to 10(4) s(-1). This conformational exchange is supposed to be due to an interaction between two α-helixes of (1-71)bacterioopsin, since the hydrolysis of the peptide bond in the loop region results in the disappearance of exchange line broadening. (15)N relaxation rates and (1)H-(15)N NOE values were interpreted using the model-free approach of Lipari and Szabo [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559]. In addition to overall rotation of the molecule, the backbone N-H vectors of the peptides are involved in two types of internal motions: fast, on a time scale <20 ps, and intermediate, on a time scale close to 1 ns. The intermediate dynamics in the α-helical stretches was mostly attributed to bending motions. A decrease in the order parameter of intermediate motions was also observed for residues next to Pro(50), indicating an anisotropy of the overall rotational diffusion of the molecule. Distinctly mobile regions are identified by a large decrease in the order parameter of intermediate motions and correspond to the N- and C-termini, and to a loop connecting the α-helixes of (1-71)bacterioopsin. The internal dynamics of the α-helixes on the millisecond and nanosecond time scales should be

  6. Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis.

    PubMed

    Tanio, Michikazu; Tanaka, Rikou; Tanaka, Takeshi; Kohno, Toshiyuki

    2009-03-15

    Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the 15N-AATS-labeled protein, the transformed B. choshinensis was cultured in 15N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a 15N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The 15N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.

  7. Carbon-13, sup 15 N, and sup 31 P NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans

    SciTech Connect

    Pust, S.; Vervoort, J.; Decker, K.; Bacher, A.; Mueller, F. )

    1989-01-24

    The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by {sup 13}C, {sup 15}N and {sup 31}P NMR techniques. The FAD prosthetic group was selectively enriched in {sup 13}C and {sup 15}N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells. In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment. The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atom strongly. It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor. On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system. It can unambiguously be concluded from the chemical shift of the N(1) atom that the reduced flavin is anionic. The doublet character of the N(3) and N(5) resonances suggests that bulk water has no access to the active center. The strong downfield shift of the N(1) position indicates that this atom is embedded in a polar environment, but it does not indicate the presence of a positively charged residue. The {sup 31}P NMR spectra show that the resonances of the pyrophosphate group of the bound FAD differ slightly from those of free FAD. Besides the {sup 31}P resonances from FAD, four peaks around 0 ppm are observed that belongs to bound phosphorus residues. The residues are not located close to the isoalloxazine ring.

  8. Feeding level and individual metabolic rate affect delta 13C and delta 15N values in carp: implications for food web studies.

    PubMed

    Gaye-Siessegger, Julia; Focken, Ulfert; Muetzel, Stefan; Abel, Hansjörg; Becker, Klaus

    2004-01-01

    Stable isotope analyses are often used to calculate relative contributions of multiple food sources in an animal's diet. One prerequisite for a precise calculation is the determination of the diet-tissue fractionation factor. Isotopic ratios in animals are not only affected by the composition of the diet, but also by the amount of food consumed. Previous findings regarding the latter point are controversial. As stable isotope analyses have often been used to investigate aquatic food webs, an experiment with carp (Cyprinus carpioL.) was carried out to test the influence of the feeding level and individual metabolic rate on delta(13)C and delta(15)N values of the whole body. After an initial phase, 49 carp were assigned randomly to four groups and fed the same diet at different levels for 8 weeks. For 15 fish, the energy budget was determined by indirect calorimetry. Feed and individual fish were analysed for their proximate composition, gross energy content and delta(13)C and delta(15)N values. delta(13)C and delta(15)N values differed significantly at different feeding levels. While delta(13)C values of the lipids and delta(15)N values decreased with increasing feeding rate, delta(13)C values of the lipid-free matter showed a non-linear pattern. Data obtained from fish held in the respirometric system revealed a relationship between delta(13)C values and the percentage retention of metabolizable energy. Our results show that reconstructing the diets of fish from the isotopic ratios when the feeding level and individual metabolic rates are unknown would introduce an error into the data used for back-calculation of up to 1 per thousand for both delta(13)C and delta(15)N values and may have substantial effects on the results of calculated diets. As other workers have pointed out, the development and application of stable isotopes to nutritional ecology studies is a field in its infancy and gives rise to erroneous, misleading results without nutritional, physiological

  9. Long-term 15N tracking from biological N fixation across different plant and humus components of the boreal forest

    NASA Astrophysics Data System (ADS)

    Arroniz-Crespo, Maria; Jones, David L.; Zackrisson, Olle; Nilsson, Marie-Charlotte; DeLuca, Thomas H.

    2014-05-01

    Biological N2 fixation by cyanobacteria associated with feather mosses is an important cog in the nitrogen (N) cycle of boreal forests; still, our understanding of the turnover and fate of N fixed by this association remains greatly incomplete. The 15N signature of plants and soil serves as a powerful tool to explore N dynamics in forest ecosystems. In particular, in the present study we aimed to investigate the contribution of N2 fixation to δ15N signatures of plants and humus component of the boreal forest. Here we present results from a long-term (7 years) tacking of labelled 15N2 across the humus layer, seedlings of the tree species Pinus sylvestris, two common dwarf shrub species (Empetrum hermaphroditum and Vaccinium vitis-idaea) and the feather moss Pleurozium schreibery. The enriched experiment was conducted in 2005 in a natural boreal forest in northern Sweden. Two different treatments (10% 15N2 headspace enrichment and control) were setup in nine different plots (0.5 x 0.5 m) within the forest. We observed a significant reduction of δ15N signature of the 15N-enriched moss that could be explained by a growth dilution effect. Nevertheless, after 5 years since 15N2 enrichment some of the label 15N was still detected on the moss and in particular in the dead tissue. We could not detect a clear transfer of the labelled 15N2 from the moss-cyanobacteria system to other components of the ecosystem. However, we found consistence relationship through time between increments of δ15N signature of some of the forest components in plots which exhibited higher N fixation rates in the moss. In particular, changes in natural abundance δ15N that could be associated with N fixation were more apparent in the humus layer, the dwarf shrub Vaccinium vitis-idaea and the pine seedlings when comparing across plots and years.

  10. The acyl nitroso Diels-Alder (ANDA) reaction of sorbate derivatives: an X-ray and 15N NMR study with an application to amino-acid synthesis.

    PubMed

    Bollans, Lee; Bacsa, John; Iggo, Jonathan A; Morris, Gareth A; Stachulski, Andrew V

    2009-11-07

    We present a study of the acyl nitroso Diels-Alder (ANDA) reaction of sorbate esters and sorbic alcohol derivatives, using alkoxycarbonyl nitroso dienophiles. An optimisation of the reaction conditions for ethyl sorbate is first presented, and the product is used in an efficient synthesis of 5-methylornithine. Structure-reactivity trends in sorbic alcohol (E,E-2,4-hexadien-1-ol) and its acylated analogues are then discussed. We present single-crystal X-ray structural proof for key adducts in both series and present in detail a novel HMBC/HSQC ((1)H-(15)N) criterion for ready distinction of regioisomers arising from such ANDA reactions.

  11. Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

    PubMed

    Gaudin, Zachary; Cerveau, Delphine; Marnet, Nathalie; Bouchereau, Alain; Delavault, Philippe; Simier, Philippe; Pouvreau, Jean-Bernard

    2014-01-21

    An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.

  12. The 15N isotope to evaluate fertilizer nitrogen absorption efficiency by the coffee plant.

    PubMed

    Fenilli, Tatiele A B; Reichart, Klaus; Bacchi, Osny O S; Trivelin, Paulo C O; Dourado-Neto, Durval

    2007-12-01

    The use of the 15N label for agronomic research involving nitrogen (N) cycling and the fate of fertilizer-N is well established, however, in the case of long term experimentation with perennial crops like citrus, coffee and rubber tree, there are still shortcomings mainly due to large plant size, sampling procedures, detection levels and interferences on the system. This report tries to contribute methodologically to the design and development of 15N labeled fertilizer experiments, using as an example a coffee crop fertilized with 15N labeled ammonium sulfate, which was followed for two years. The N of the plant derived from the fertilizer was studied in the different parts of the coffee plant in order to evaluate its distribution within the plant and the agronomic efficiency of the fertilizer application practice. An enrichment of the fertilizer-N of the order of 2% 15N abundance was sufficient to study N absorption rates and to establish fertilizer-N balances after one and two years of coffee cropping. The main source of errors in the estimated values lies in the inherent variability among field replicates and not in the measurements of N contents and 15N enrichments of plant material by mass-spectrometry.

  13. Nitrate turnover in a peat soil under drained and rewetted conditions: results from a [(15)N]nitrate-bromide double-tracer study.

    PubMed

    Russow, Rolf; Tauchnitz, Nadine; Spott, Oliver; Mothes, Sibylle; Bernsdorf, Sabine; Meissner, Ralph

    2013-01-01

    Under natural conditions, peatlands are generally nitrate-limited. However, recent concerns about an additional N input into peatlands by atmospheric N deposition have highlighted the risk of an increased denitrification activity and hence the likelihood of a rise of emissions of the greenhouse gas nitrous oxide. Therefore, the aim of the present study was to investigate the turnover of added nitrate in a drained and a rewetted peatland using a [(15)N]nitrate-bromide double-tracer method. The double-tracer method allows a separation between physical effects (dilution, dispersion and dislocation) and microbial and chemical nitrate transformation by comparing with the conservative Br(-) tracer. In the drained peat site, low NO3(-) consumption rates have been observed. In contrast, NO3(-) consumption at the rewetted peat site rises rapidly to about 100% within 4 days after tracer application. Concomitantly, the (15)N abundances of nitrite and ammonium in soil water increased and lead to the conclusion that, besides commonly known NO3(-) reduction to nitrite (i.e. denitrification), a dissimilatory nitrate reduction to ammonium has simultaneously taken place. The present study reveals that increasing NO3(-) inputs into rewetted peatlands via atmospheric deposition results in a rapid NO3(-) consumption, which could lead to an increase in N2O emissions into the atmosphere.

  14. sup 13 C and sup 15 N NMR studies on the interaction between 6,7-dimethyl-8-ribityllumazine and lumazine protein

    SciTech Connect

    Vervoort, J.; Mueller, F. ); O'Kane, D.J.; Lee, J. ); Bacher, A.; Strobl, G. )

    1990-02-20

    The interaction between the prosthetic group 6,7-dimethyl-8-(1{prime}-D-ribityl)lumazine and the lumazine apoproteins from two marine bioluminescent bacteria, one from a relatively thermophilic species, Photobacterium leiognathi, and the other from a psychrophilic species, Photobacterium phosphoreum, was studied by {sup 13}C and {sup 15}N NMR using various selectively enriched derivatives. It is shown that the electron distribution in the protein-bound 6,7-dimethyl-8-ribityllumazine differs from that of free 6,7-dimethyl-8-ribityllumazine in buffer. The {sup 13}C and {sup 15}N chemical shifts indicate that the protein-bound 6,7-dimethyl-8-ribityllumazine is embedded in a polar environment and that the ring system is strongly polarized. It is concluded that the two carbonyl groups play an important role in the polarization of the molecule. The N(3)-H group is not accessible to bulk solvent. The N(8) atom is sp{sup 2} hybridized and has {delta}+ character. Nuclear Overhauser effect studies indicate that the 6,7-dimethyl-8-ribityllumazine ring is rigidly bound with no internal mobility. The NMR results indicate that the interaction between the ring system and the two apoproteins is almost the same.

  15. (15)N-ammonium and (15)N-nitrate uptake of a 15-year-old Picea abies plantation.

    PubMed

    Buchmann, N; Schulze, E-D; Gebauer, G

    1995-06-01

    Throughfall nitrogen of a 15-year-old Picea abies (L.) Karst. (Norway spruce) stand in the Fichtelgebirge, Germany, was labeled with either (15)N-ammonium or (15)N-nitrate and uptake of these two tracers was followed during two successive growing seasons (1991 and 1992). (15)N-labeling (62 mg (15)N m(-2) under conditions of 1.5 g N m(-2) atmospheric nitrogen deposition) did not increase N concentrations in plant tissues. The (15)N recovery within the entire stand (including soils) was 94%±6% of the applied (15)N-ammonium tracer and 100%±6% of the applied (15)N-nitrate tracer during the 1st year of investigation. This decreased to 80%±24% and 83%±20%, respectively, during the 2nd year. After 11 days, the (15)N tracer was detectable in 1-year-old spruce needles and leaves of understory species. After 1 month, tracer was detectable in needle litter fall. At the end of the first growing season, more than 50% of the (15)N taken up by spruce was assimilated in needles, and more than 20% in twigs. The relative distribution of recovered tracer of both (15)N-ammonium and (15)N-nitrate was similar within the different foliage age classes (recent to 11-year-old) and other compartments of the trees. (15)N enrichment generally decreased with increasing tissue age. Roots accounted for up to 20% of the recovered (15)N in spruce; no enrichment could be detected in stem wood. Although (15)N-ammonium and (15)N-nitrate were applied in the same molar quantities ((15)NH 4(+) : (15)NO 3(-) =1:1), the tracers were diluted differently in the inorganic soil N pools ((15)NH 4(+) /NH 4(+) : (15)NO 3(-) /NO 3(-) =1:9). Therefore the measured (15)N amounts retained by the vegetation do not represent the actual fluxes of ammonium and nitrate in the soil solution. Use of the molar ammonium-to-nitrate ratio of 9:1 in the soil water extract to estimate (15)N uptake from inorganic N pools resulted in a 2-4 times higher ammonium than nitrate uptake by P. abies.

  16. Heteronuclear NMR studies of cobalamins. 11. sup 15 N NMR studies of the axial nucleotide and amide side chains of cyanocobalamin and dicyanocobamides

    SciTech Connect

    Brown, K.; Brooks, H.B.; Xiang, Zou ); Victor, M.; Ray, A. ); Timkovich, R. )

    1990-11-28

    Spectroscopic and thermodynamic evidence for the structure of cobalamines and dicyanocobalamin (CN){sub 2}Cbl have been previously reported. The structure indicated the occurrence of the so-called tuck-in species. Further observations and characterization of the tuck-in species of (CN){sub 2}Cbl by {sup 15}N NMR spectroscopy are presented herein. These results represent the first observation of the {sup 15}N NMR spectrum of benzimidazole nucleotide of cobalamins. The first NMR observation of the amide protons of cobalamins and their connectivity to the amide nitrogens are also reported. 50 refs., 2 figs., 2 tabs.

  17. Biogeochemical Indicators in High- and Low-Arctic Marine and Terrestrial Avian Community Changes: Comparative Isotopic (13C, 15N, and 34S) Studies in Alaska and Greenland

    NASA Astrophysics Data System (ADS)

    Causey, D.; Bargmann, N. A.; Burnham, K. K.; Burnham, J. L.; Padula, V. M.; Johnson, J. A.; Welker, J. M.

    2011-12-01

    Understanding the complex dynamics of environmental change in northern latitudes is of paramount importance today, given documented rapid shifts in sea ice, plant phenology, temperatures, deglaciation, and habitat fidelity. This knowledge is particularly critical for Arctic avian communities, which are integral components by which biological teleconnections are maintained between the mid and northern latitudes. Furthermore, Arctic birds are fundamental to Native subsistence lifestyles and a focus for conservation activities. Avian communities of marine and terrestrial Arctic environments represent a broad spectrum of trophic levels, from herbivores (eg., geese Chen spp.), planktivores (eg., auklets Aethia spp.), and insectivores (eg., passerines: Wheatears Oenanthe spp., Longspurs Calcarius spp.), to predators of marine invertebrates (eg., eiders Somateria spp.), nearshore and offshore fish (eg., cormorants Phalacrocorax spp, puffins Fratercula spp.), even other bird species (eg., gulls Larus spp., falcons Peregrinus spp.). This diversity of trophic interconnections is an integral factor in the dynamics of Arctic ecosystem ecology, and they are key indicators for the strength and trajectories of change. We are especially interested in their feeding ecology, using stable isotope-diet relations to examine historical diets and to predict future feeding ecology by this range of species. Since 2009, we have been studying the foodweb ecology using stable isotopes (δ13C, δ15N, δ34S) of contemporaneous coastal and marine bird communities in High Arctic (Northwest Greenland) and Low Arctic (western Aleutian Islands, AK). We are quantifying the isotopic values of blood, organ tissues, and feathers, and have carried out comparisons between native and lipid-extracted samples. Although geographically distant, these communities comprise similar taxonomic and ecological congeners, including several species common to both (eg., Common Eider, Black-legged Kittiwake, Northern

  18. A study of pyridyl nitrosyl iron(II) tetraphenyl 15N4-porphyrin. NO geometry and spin coupling to the pyrrole nitrogens

    NASA Astrophysics Data System (ADS)

    Gilbert, D. C.; Dikanov, S. A.; Doetschman, D. C.; Smeija, J. A.

    1999-12-01

    Spin coupling with pyrrole nitrogens and NO geometry in pyridyl-NO-Fe(II) tetraphenyl- 15N4-porphyrin, examined with hyperfine sublevel correlation spectroscopy (HYSCORE), was studied because of renewed interest in diatomic molecule bound ferrous hemes, e.g. the physiologically important NO synthase. Dipolar coupling locates the effective electron spin position (0.109±0.008 nm from the ring center, 0.106±0.006 nm above the ring plane and projecting 37±2° from the nearest pyrrole nitrogen). The NO projection in an X-ray study of the 4-methyl piperidine complex is 38.6°. The negative pyrrole nitrogen spin densities induced by the NO obey a sinusoidal angular relationship.

  19. 13C and 15N NMR studies of iron-bound cyanides of heme proteins and related model complexes: sensitive probe for detecting hydrogen-bonding interactions at the proximal and distal sides.

    PubMed

    Fujii, Hiroshi; Yoshida, Tadashi

    2006-08-21

    Studies of the 13C and 15N NMR paramagnetic shifts of the iron-bound cyanides in the ferric cyanide forms of various heme proteins containing the proximal histidine and related model complexes are reported. The paramagnetic shifts of the 13C and 15N NMR signals of the iron-bound cyanide are not significantly affected by the substitution of the porphyrin side chains. On the other hand, the paramagnetic shifts of both the 13C and 15N NMR signals decrease with an increase in the donor effect of the proximal ligand, and the 13C NMR signal is more sensitive to a modification of the donor effect of the proximal ligand than the 15N NMR signal. With the tilt of the iron-imidazole bond, the paramagnetic shift of the 13C NMR signal increases, whereas that of the 15N NMR signal decreases. The hydrogen-bonding interaction of the iron-bound cyanide with a solvent decreases the paramagnetic shift of both 13C and 15N NMR signals, and the effect is more pronounced for the 15N NMR signal. Data on the 13C and 15N NMR signals of iron-bound cyanide for various heme proteins are also reported and analyzed in detail. Substantial differences in the 13C and 15N NMR shifts for the heme proteins can be explained on the basis of the results for the model complexes and structures around the heme in the heme proteins. The findings herein show that the paramagnetic shift of the 13C NMR signal of the iron-bound cyanide is a good probe to estimate the donor effect of the proximal imidazole and that the ratio of 15N/13C NMR shifts allows the hydrogen-bonding interaction on the distal side to be estimated.

  20. Study of the 15N(p,n)15O reaction as a monoenergetic neutron source for the measurement of differential scattering cross sections

    NASA Astrophysics Data System (ADS)

    Poenitz, E.; Nolte, R.; Schmidt, D.; Chen, G.

    2017-03-01

    The 15N(p,n) reaction is a promising candidate for the production of monoenergetic neutrons with energies of up to 5.7 MeV at the facilities where the T(p,n)3He reaction cannot be used. The characteristic properties of this reaction were studied focusing on its suitability as a source of monoenergetic neutrons for the measurement of differential scattering cross sections in the neutron energy range of 2 MeV to 5 MeV . For this purpose differential and integral cross sections were measured and the choice of optimum target conditions was investigated. The reaction has already been used successfully to measure of elastic and inelastic neutron scattering cross sections for natPb in the energy range from 2 MeV to 4 MeV and for 209Bi and 181Ta at 4 MeV .

  1. Complexity of the food web structure of the Ascophyllum nodosum zone evidenced by a δ13C and δ15N study

    NASA Astrophysics Data System (ADS)

    Golléty, Claire; Riera, Pascal; Davoult, Dominique

    2010-10-01

    Rocky shores dominated by canopy-forming macroalgae are characterized by complex communities making it difficult to assess whether the most abundant primary producers are at the base of the food web. This difficulty is exacerbated by the seasonal- and regional-scale variations of environmental and biotic factors that can affect the main trophic pathways. The food web structure of the Ascophyllum nodosum zone was studied during three seasons and at two sites separated by several 100s of kilometers by measuring the δ13C and δ15N of the major food sources and the dominant consumers of the zone. Despite the variability in isotopic compositions, both sites underwent similar significant seasonal variations. The main primary producers of the zone, A.nodosum, Fucus vesiculosus and Fucus serratus, were not at the base of the main trophic pathway but part of the diverse number of basal resources supporting the food web. The use of community-wide metric indices allowed further defining the food web structure of the A. nodosum zone as one characterized by trophic redundancy and numerous major trophic pathways. Indeed, grazers were dominated by generalists, filter-feeders utilized both planktonic and benthic organic matter, and predators displayed a high degree of omnivory. The range of values in δ15N showed a high spatiotemporal variability within and an important overlap between trophic groups. This prevented establishing distinctive trophic levels and further emphasized the complexity of the food web structure. The spatiotemporal stability of the relative isotopic composition of the dominant consumers within trophic groups and the low variability of the community-wide indices suggested a stability of the food web structure of the A.nodosum zone at a regional scale.

  2. Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR, (14,15)N HYSCORE, and DFT Study.

    PubMed

    Rendon, Julia; Biaso, Frédéric; Ceccaldi, Pierre; Toci, René; Seduk, Farida; Magalon, Axel; Guigliarelli, Bruno; Grimaldi, Stéphane

    2017-03-31

    Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in (98)Mo and (15)N nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively (15)N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.

  3. Diffusion technique for 15N and inorganic N analysis of low-N aqueous solutions and Kjeldahl digests.

    PubMed

    Chen, Rui Rui; Dittert, Klaus

    2008-06-01

    Diffusion of ammonia is a common sample preparation method for the stable isotope analysis of inorganic nitrogen in aqueous solution. Classical diffusion methods usually require 6-12 days of diffusion and often focus on (15)N/(14)N analysis only. More recent studies have discussed whether complete N recovery was necessary for the precise analysis of stable N isotope ratios. In this paper we present a newly revised diffusion technique that allows correct and simultaneous determination of total N and (15)N at% from aqueous solutions and Kjeldahl digests, with N concentrations down to sub-0.5-mg N L(-1) levels, and it is tested under different conditions of (15)N isotope labelling. With the modification described, the diffusion time was reduced to 72 h, while the ratios of measured and expected (15)N at% were greater than 99% and the simultaneous recovery of total N was >95%. Analysis of soil microbial biomass N and its (15)N/(14)N ratio is one of the most important applications of this diffusion technique. An experiment with soil extracts spiked with (15)N-labelled yeast showed that predigestion was necessary to prevent serious N loss during Kjeldahl digestion of aqueous samples (i.e. soil extracts). The whole method of soil microbial biomass N preparation for (15)N/(14)N analysis included chloroform fumigation, predigestion, Kjeldahl digestion and diffusion. An experiment with soil spiked with (15)N-labelled yeast was carried out to evaluate the method. Results showed a highly significant correlation of recovered and added N, with the same recovery rate (0.21) of both total N and (15)N. A k(N) value of 0.25 was obtained based on the data. In conclusion, the diffusion method works for soil extracts and microbial biomass N determination and hence could be useful in many types of soil/water studies.

  4. Development and application of 15N-tracer substances for measuring the whole-body protein turnover rates in the human, especially in neonates: a review.

    PubMed

    Wutzke, Klaus D

    2012-06-01

    Our research group of the Children's Hospital of the University of Rostock (Rostock group) has long-time experience in (15)N-labelling and in using yeast protein and its hydrolysates for tracer kinetic studies to evaluate parameters of the whole-body protein metabolism in premature infants. The particular advantage of applying an economically convenient, highly (15)N-enriched, and completely labelled yeast protein for evaluating protein turnover rates is the fact that the (15)N dose is spread among all proteinogenic amino acids. The absorption has been improved by hydrolysing [(15)N]yeast protein with thermitase into a mixture of amino acids, dipeptides and tripeptides so that faecal analysis becomes unnecessary when determining turnover rates. The review shows that, in contrast to the application of single (15)N-labelled amino acids with resulting overestimation of protein turnover rates, the (15)N-labelled yeast protein thermitase hydrolysate represents the amino acid metabolism more closely without causing amino acid imbalances. The (15)N-labelled yeast protein thermitase hydrolysate leads to the estimation of reliable protein turnover rates, particularly in premature infants.

  5. Compound-specific 15N analysis of amino acids in 15N tracer experiments provide an estimate of newly synthesised soil protein from inorganic and organic substrates

    NASA Astrophysics Data System (ADS)

    Charteris, Alice; Michaelides, Katerina; Evershed, Richard

    2015-04-01

    Organic N concentrations far exceed those of inorganic N in most soils and despite much investigation, the composition and cycling of this complex pool of SOM remains poorly understood. A particular problem has been separating more recalcitrant soil organic N from that actively cycling through the soil system; an important consideration in N cycling studies and for the soil's nutrient supplying capacity. The use of 15N-labelled substrates as stable isotope tracers has contributed much to our understanding of the soil system, but the complexity and heterogeneity of soil organic N prevents thorough compound-specific 15N analyses of organic N compounds and makes it difficult to examine any 15N-labelled organic products in any detail. As a result, a significant proportion of previous work has either simply assumed that since the majority of soil N is organic, all of the 15N retained in the soil is organic N (e.g. Sebilo et al., 2013) or subtracted 15N-labelled inorganic compounds from bulk values (e.g. Pilbeam et al., 1997). While the latter approach is more accurate, these methods only provide an estimate of the bulk 15N value of an extremely complex and non-uniformly labelled organic pool. A more detailed approach has been to use microbial biomass extraction (Brookes et al., 1985) and subsequent N isotopic analysis to determine the 15N value of biomass-N, representing the fraction of 15N assimilated by microbes or the 15N cycling through the 'living' or 'active' portion of soil organic N. However, this extraction method can only generate estimates and some lack of confidence in its validity and reliability remains. Here, we present an alternative technique to obtain a measure of the assimilation of an applied 15N substrate by the soil microbial biomass and an estimate of the newly synthesized soil protein, which is representative of the magnitude of the active soil microbial biomass. The technique uses a stable isotope tracer and compound-specific 15N analysis, but

  6. Mechanism of Solid-State Thermolysis of Ammonia Boraine: 15N NMR Study Using Fast Magic-Angle Spinning and Dynamic Nuclear Polarization

    SciTech Connect

    Kobayashi, Takeshi; Gupta, Shalabh; Caporini, Marc A; Pecharsky, Vitalij K; Pruski, Marek

    2014-08-28

    The solid-state thermolysis of ammonia borane (NH3BH3, AB) was explored using state-of-the-art 15N solid-state NMR spectroscopy, including 2D indirectly detected 1H{15N} heteronuclear correlation and dynamic nuclear polarization (DNP)-enhanced 15N{1H} cross-polarization experiments as well as 11B NMR. The complementary use of 15N and 11B NMR experiments, supported by density functional theory calculations of the chemical shift tensors, provided insights into the dehydrogenation mechanism of AB—insights that have not been available by 11B NMR alone. Specifically, highly branched polyaminoborane derivatives were shown to form from AB via oligomerization in the “head-to-tail” manner, which then transform directly into hexagonal boron nitride analog through the dehydrocyclization reaction, bypassing the formation of polyiminoborane.

  7. Uptake and Reduction of [15N]Nitrate by Intact Soybean Plants in the Dark

    PubMed Central

    Nicholas, Joseph C.; Harper, James E.

    1985-01-01

    Experiments were conducted to determine if nitrate (15N-labeled) was taken up and assimilated by intact soybean (Glycine max [L.] Merr. cv Williams) plants during extended periods of dark. Nitrate was taken up by soybean roots throughout a 12-hour dark period. The 15N-labeled nitrogen was also translocated to the plant shoots, but at a slower rate than the rate of accumulation in the roots. Much of the nitrogen (15N-labeled) was present in a nonreduced form, although considerable soluble-reduced nitrogen (15N-labeled) accumulated throughout the dark period. The 15N-labeled, soluble-reduced nitrogen fraction accounted for nearly 30% of the total 15N found in plant roots and more than 63% of the total 15N found in plant tops after 12 hours of dark. This provided evidence that intact soybean plants take up and metabolize significant quantities of nitrate to reduced N forms in the dark. In addition to nitrate influx during the dark, it was shown that there was a concomitant loss of 15N-labeled nitrogen compounds from previously 15N-labeled plants to a natural abundance 15N nutrient solution. Thus, evidence was obtained which indicated that light was not directly essential for flux and reduction of nitrate by intact soybean plants. PMID:16664059

  8. Efficient Synthesis of Nicotinamide-1-15N for Ultrafast NMR Hyperpolarization Using Parahydrogen

    PubMed Central

    2016-01-01

    Nicotinamide (a vitamin B3 amide) is one of the key vitamins as well as a drug for treatment of M. tuberculosis, HIV, cancer, and other diseases. Here, an improved Zincke reaction methodology is presented allowing for straightforward and scalable synthesis of nicotinamide-1-15N with an excellent isotopic purity (98%) and good yield (55%). 15N nuclear spin label in nicotinamide-1-15N can be NMR hyperpolarized in seconds using parahydrogen gas. NMR hyperpolarization using the process of temporary conjugation between parahydrogen and to-be-hyperpolarized biomolecule on hexacoordinate iridium complex via the Signal Amplification By Reversible Exchange (SABRE) method significantly increases detection sensitivity (e.g., >20 000-fold for nicotinamide-1-15N at 9.4 T) as has been shown by Theis T. et al. (J. Am. Chem. Soc.2015, 137, 1404), and hyperpolarized in this fashion, nicotinamide-1-15N can be potentially used to probe metabolic processes in vivo in future studies. Moreover, the presented synthetic methodology utilizes mild reaction conditions, and therefore can also be potentially applied to synthesis of a wide range of 15N-enriched N-heterocycles that can be used as hyperpolarized contrast agents for future in vivo molecular imaging studies. PMID:26999571

  9. Nitrogen input 15N signatures are reflected in plant 15N natural abundances in subtropical forests in China

    NASA Astrophysics Data System (ADS)

    Abdisa Gurmesa, Geshere; Lu, Xiankai; Gundersen, Per; Fang, Yunting; Mao, Qinggong; Hao, Chen; Mo, Jiangming

    2017-05-01

    Natural abundance of 15N15N) in plants and soils can provide time-integrated information related to nitrogen (N) cycling within ecosystems, but it has not been well tested in warm and humid subtropical forests. In this study, we used ecosystem δ15N to assess effects of increased N deposition on N cycling in an old-growth broad-leaved forest and a secondary pine forest in a high-N-deposition area in southern China. We measured δ15N of inorganic N in input and output fluxes under ambient N deposition, and we measured N concentration (%N) and δ15N of major ecosystem compartments under ambient deposition and after decadal N addition at 50 kg N ha-1yr-1, which has a δ15N of -0.7 ‰. Our results showed that the total inorganic N in deposition was 15N-depleted (-10 ‰) mainly due to high input of strongly 15N-depleted NH4+-N. Plant leaves in both forests were also 15N-depleted (-4 to -6 ‰). The broad-leaved forest had higher plant and soil %N and was more 15N-enriched in most ecosystem compartments relative to the pine forest. Nitrogen addition did not significantly affect %N in the broad-leaved forest, indicating that the ecosystem pools are already N-rich. However, %N was marginally increased in pine leaves and significantly increased in understory vegetation in the pine forest. Soil δ15N was not changed significantly by the N addition in either forest. However, the N addition significantly increased the δ15N of plants toward the 15N signature of the added N, indicating incorporation of added N into plants. Thus, plant δ15N was more sensitive to ecosystem N input manipulation than %N in these subtropical forests. We interpret the depleted δ15N of plants as an imprint from the high and 15N-depleted N deposition that may dominate the effects of fractionation that are observed in most warm and humid forests. Fractionation during the steps of N cycling could explain the difference between negative δ15N in plants and positive δ15N in soils, and the increase

  10. Complete Thermodynamic Characterization of the Multiple Protonation Equilibria of the Aminoglycoside Antibiotic Paromomycin: A Calorimetric and Natural Abundance 15N NMR Study

    PubMed Central

    Barbieri, Christopher M.; Pilch, Daniel S.

    2006-01-01

    The binding of aminoglycoside antibiotics to a broad range of macromolecular targets is coupled to protonation of one or more of the amino groups that typify this class of drugs. Determining how and to what extent this linkage influences the energetics of the aminoglycoside-macromolecule binding reaction requires a detailed understanding of the thermodynamics associated with the protonation equilibria of the aminoglycoside amino groups. In recognition of this need, a calorimetric- and NMR-based approach for obtaining the requisite thermodynamic information is presented using paromomycin as the model aminoglycoside. Temperature- and pH-dependent 15N NMR studies provide pKa values for the five paromomycin amino groups, as well as the temperature dependence of these pKa values. These studies also indicate that the observed pKa values associated with the free base form of paromomycin are lower in magnitude than the corresponding values associated with the sulfate salt form of the drug. This difference in pKa is due to drug interactions with the sulfate counterions at the high drug concentrations (≥812 mM) used in the 15N NMR studies. Isothermal titration calorimetry studies conducted at drug concentrations ≤45 μM reveal that the extent of paromomycin protonation linked to the binding of the drug to its pharmacologically relevant target, the 16 S rRNA A-site, is consistent with the pKa values of the free base and not the sulfate salt form of the drug. Temperature- and pH-dependent isothermal titration calorimetry studies yield exothermic enthalpy changes (ΔH) for protonation of the five paromomycin amino groups, as well as positive heat capacity changes (ΔCp) for three of the five amino groups. Regarded as a whole, the results presented here represent an important first step toward establishing a thermodynamic database that can be used to predict how aminoglycoside-macromolecule binding energetics will be influenced by conditions such as temperature, pH, and

  11. Soil N transformations and its controlling factors in temperate grasslands in China: A study from 15N tracing experiment to literature synthesis

    NASA Astrophysics Data System (ADS)

    Wang, Jing; Wang, Liang; Feng, Xiaojuan; Hu, Huifeng; Cai, Zucong; Müller, Christoph; Zhang, Jinbo

    2016-12-01

    Temperate grasslands in arid and semiarid regions cover about 40% of the total land area in China. So far, only a few studies have studied the N transformations in these important ecosystems. In the present study, soil gross N transformation rates in Inner Mongolia temperate grasslands in China were determined using a 15N tracing experiment and combined with a literature synthesis to identify the soil N transformation characteristics and their controlling factors in a global perspective. Our results showed that the rates of gross N mineralization and immobilization NH4+ were significantly lower, while autotrophic nitrification rates were significantly higher in Chinese temperate grassland soils compared to other regions in the world. In particular, the primary mineral N consumption processes, i.e., immobilization of NO3- and NH4+, and dissimilatory nitrate reduction to ammonium, were on average much lower in temperate grassland soils in China, compared to other temperate grassland regions. The reduced heterotrophic activity and microbial growth associated with lower soil organic carbon and arid climate (e.g., mean annual precipitation) were identified as the main factors regulating soil N cycling in the studied regions in China. To restrict NO3- accumulation and associated high risks of N losses in these arid and semiarid ecosystems in China, it is important to develop the regimes of soil organic C and water management that promote the retention of N in these grassland ecosystems.

  12. 1H, 15N and 13C NMR Assignments of Mouse Methionine Sulfoxide Reductase B2

    PubMed Central

    Breivik, Åshild S.; Aachmann, Finn L.; Sal, Lena S.; Kim, Hwa-Young; Del Conte, Rebecca; Gladyshev, Vadim N.; Dikiy, Alexander

    2011-01-01

    A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with 15N and 15N/13C was generated. We report here the 1H, 15N and 13C NMR assignments of the reduced form of this protein. PMID:19636904

  13. The First in Vivo Observation of 13C- 15N Coupling in Mammalian Brain

    NASA Astrophysics Data System (ADS)

    Kanamori, Keiko; Ross, Brian D.

    2001-12-01

    [5-13C,15N]Glutamine, with 1J(13C-15N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by 13C MRS at 4.7 T. The brain [5-13C]glutamine peak consisted of the doublet from [5-13C,15N]glutamine and the center [5-13C,14N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-13C,15N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-13C,15N]glutamine was formed by glial uptake of released neurotransmitter [5-13C]glutamate and its reaction with 15NH3 catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively13C-enriched by intravenous [2,5-13C]glucose infusion to 13C-label whole-brain glutamate C5, followed by [12C]glucose infusion to chase 13C from the small and rapidly turning-over glial glutamate pool, leaving 13C mainly in the neurotransmitter [5-13C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-13C,15N]glutamine arises from a coupling between 13C of neuronal origin and 15N of glial origin. Measurement of the rate of brain [5-13C,15N]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.

  14. AGB fluorine nucleosynthesis studied by means of Trojan-horse method: the case of {sup 15}N(p,{alpha}){sup 12}C

    SciTech Connect

    Pizzone, R. G.; Spitaleri, C.; La Cognata, M.; Cherubini, S.; Crucilla, V.; Gulino, M.; Lamia, L.; Puglia, S. M. R.; Rapisarda, G. G.; Romano, S.; Sergi, S.; Tumino, A.; Tribble, R.; Trache, L.; Fu, C.; Goldberg, V.; Mukhamedzhanov, A.; Tabacaru, G.

    2008-05-12

    The low-energy bare-nucleus cross section for {sup 15}N(p,{alpha}){sup 12}C is extracted by means of the Trojan-horse Method applied to the {sup 2}H({sup 15}N,{alpha}){sup 12}C)n reaction at E{sub beam} = 60 MeV. The astrophysical S-factor is deduced and compared to the direct data in the same energy region. A fair agreement with direct data down to 80 keV is found if energy resolution effects are taken into account.

  15. Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study.

    PubMed

    Hein, Christopher; Löhr, Frank; Schwarz, Daniel; Dötsch, Volker

    2017-05-01

    Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial (15) N-, (13) C(α) -, and (13) C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves (15) N, 1-(13) C, 2-(13) C, fully (15) N/(13) C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide (1) H and (15) N resonances need to be obtained, even in cases where sensitivity is the limiting factor. © 2016 Wiley Periodicals, Inc.

  16. Compound-specific 15N stable isotope probing of N assimilation by the soil microbial biomass: a new methodological paradigm in soil N cycling

    NASA Astrophysics Data System (ADS)

    Charteris, A. F.; Knowles, T. D. J.; Michaelides, K.; Evershed, R. P.

    2015-10-01

    A compound-specific nitrogen-15 stable isotope probing (15N-SIP) technique is described which allows investigation of the fate of inorganic- or organic-N amendments to soils. The technique uses gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the δ15N values of individual amino acids (AAs; determined as N-acetyl, O-isopropyl derivatives) as proxies of biomass protein production. The δ15N values are used together with AA concentrations to quantify N assimilation of 15N-labelled substrates by the soil microbial biomass. The utility of the approach is demonstrated through incubation experiments using inorganic 15N-labelled substrates ammonium (15NH4+) and nitrate (15NO3-) and an organic 15N-labelled substrate, glutamic acid (15N-Glu). Assimilation of all the applied substrates was undetectable based on bulk soil properties, i.e. % total N (% TN), bulk soil N isotope composition and AA concentrations, all of which remained relatively constant throughout the incubation experiments. In contrast, compound-specific AA δ15N values were highly sensitive to N assimilation, providing qualitative and quantitative insights into the cycling and fate of the applied 15N-labelled substrates. The utility of this 15N-AA-SIP technique is considered in relation to other currently available methods for investigating the microbially-mediated assimilation of nitrogenous substrates into the soil organic N pool. This approach will be generally applicable to the study of N cycling in any soil, or indeed, in any complex ecosystem.

  17. Dynamics in Thermotoga neapolitana adenylate kinase: 15N relaxation and hydrogen-deuterium exchange studies of a hyperthermophilic enzyme highly active at 30 degrees C.

    PubMed

    Krishnamurthy, Harini; Munro, Kim; Yan, Honggao; Vieille, Claire

    2009-03-31

    Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 degrees C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK's increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 degrees C by localizing flexibility to the hinge regions that are key to facilitating conformational changes.

  18. Protein-energy status and 15N-glycine kinetic study of child a cirrhotic patients fed low- to high-protein energy diets.

    PubMed

    Dichi, I; Dichi, J B; Papini-Berto, S J; Angeleli, A Y; Bicudo, M H; Rezende, T A; Burini, R C

    1996-01-01

    In five male cirrhotic patients (Child A) and in four age- and sex-matched healthy control subjects, whole-body protein turnover was measured using a single oral dose of 15N-glycine as a tracer and urinary ammonia as end product. Subjects were studied in the fasting and feeding state, with different levels of protein and energy intake. The patients were underweight and presented lower plasma transthyretin and retinol-binding protein levels. When compared with controls, the kinetic studies showed patients to be hypometabolic in the fasting (D0) state and with the control diet [D1 = (0.85 g of protein/ 154 kJ) x kg-1.day-1]. However, when corrected by body weight, the kinetic differences between groups disappeared, whereas the N-retention in the feeding state showed better results for the patients due mainly to their efficient breakdown decrease. When fed high-level protein or energy diets [D1 = (0.9 g protein/195 kJ) and D3 = (1.56 g protein/158 kJ) x kg-1.day-1], the patients showed D0 = D1 = D2 < D3 for N-flux and (D0 = D1) < D3 (D2 is intermediary) for protein synthesis. Thus, the present data suggest that the remaining mass of the undernourished mild cirrhotic patients has fairly good protein synthesis activity and also that protein, rather than energy intake, would be the limiting factor for increasing their whole-body protein synthesis.

  19. Nitrogen assimilation and short term retention in a nutrient-rich tidal freshwater marsh - a whole ecosystem 15N enrichment study

    NASA Astrophysics Data System (ADS)

    Gribsholt, B.; Struyf, E.; Tramper, A.; de Brabandere, L.; Brion, N.

    2006-07-01

    We conducted two (May 2002 and September 2003) pulse additions of 15NH4+ to the flood water inundating a tidal freshwater marsh fringing the nutrient-rich Scheldt River (Belgium) and traced the fate of ammonium in the intact ecosystem. Here we report in detail the 15N uptake into the various marsh components (leaves, roots, sediment, leaf litter and invertebrate fauna), and the 15N retention on a scale of 15 days. We particularly focus on the contributions of the rooted macrophytes and the microbial community in the sediment and on plant litter. Assimilation and short term retention of 15NH4+ was low on both occasions. Only 4-9% of the added 15N trace was assimilated, corresponding to 13-22% and 8-18% of the processed 15N (i.e. not exported as 15NH4+ in May and September, respectively. In May nitrogen assimilation rate (per hour inundated) was >3 times faster than in September. Macrophytes (above- and below ground) were of limited importance for short term 15N retention accounting for <6% of the total 15NH4+ processed by the marsh. The less dominant herbaceous species were more important (on an area basis) than the dominant reed (Phragmites australis). The microbial community colonizing the sediment and litter surfaces were responsible for most nitrogen assimilation and short-term retention in the marsh. The large reactive surface area available for microbial colonization together with direct plant uptake, are the crucial components for nitrogen assimilation, retention and transformation in nutrient-rich tidal freshwater marshes.

  20. Chlorophyll a-specific Δ14C, δ13C and δ15N values in stream periphyton: implications for aquatic food web studies

    NASA Astrophysics Data System (ADS)

    Ishikawa, N. F.; Yamane, M.; Suga, H.; Ogawa, N. O.; Yokoyama, Y.; Ohkouchi, N.

    2015-11-01

    Periphytic algae attached to a streambed substrate (periphyton) are an important primary producer in stream ecosystems. We determined the isotopic composition of chlorophyll a in periphyton collected from a stream flowing on limestone bedrock in the Seri River, central Japan. Stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) and natural radiocarbon abundances (Δ14C) were measured in chlorophyll a (δ13Cchl, δ15Nchl and Δ14Cchl) and bulk (δ13Cbulk, δ15Nbulk and Δ14Cbulk) for periphyton, a pure aquatic primary producer (Cladophora sp.) and a terrestrial primary producer (Quercus glauca). Periphyton δ13Cbulk and δ13Cchl values did not necessarily correspond to δ13Cbulk for an algal-grazing specialist (Epeorus latifolium). Periphyton Δ14Cchl values (-258 ‰ in April and -190 ‰ in October) were slightly lower than Δ14Cbulk values (-228 ‰ in April and -179 ‰ in October) but were close to the Δ14C value for dissolved inorganic carbon (DIC; -217 ± 31 ‰), which is a mixture of weathered carbonates (Δ14C = -1000 ‰), CO2 derived from aquatic and terrestrial organic matters (variable Δ14C) and dissolved atmospheric CO2 (Δ14C approximately +30 ‰ in 2013). Δ14Cchl values were also close to Δ14Cbulk for E. latifolium (-215 ‰ in April and -199 ‰ in October) and Cladophora sp. (-210 ‰), whereas the Δ14Cbulk value for Q. glauca (+27 ‰) was closer to Δ14C for atmospheric CO2. Although the bulk isotopic composition of periphyton is recognised as a surrogate for the photosynthetic algal community, natural periphyton is a mixture of aquatic and terrestrial organic materials. Our results indicate that the bulk periphyton matrix at the study site consists of 89 to 95 % algal carbon (derived from 14C-depleted DIC) and 5 to 11 % terrestrial organic carbon (derived from 14C-enriched atmospheric CO2).

  1. Chlorophyll a specific Δ14C, δ13C and δ15N values in stream periphyton: implications for aquatic food web studies

    NASA Astrophysics Data System (ADS)

    Ishikawa, N. F.; Yamane, M.; Suga, H.; Ogawa, N. O.; Yokoyama, Y.; Ohkouchi, N.

    2015-07-01

    We determined the isotopic composition of chlorophyll a in periphytic algae attached to a streambed substrate (periphyton). The samples were collected from a stream flowing on limestone bedrock in the Seri River, central Japan. Stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) and natural radiocarbon abundances (Δ14C) were measured in chlorophyll a (δ13Cchl, δ15Nchl and Δ14Cchl) and bulk (δ13Cbulk, δ15Nbulk and Δ14Cbulk) for periphyton, pure aquatic primary producer (Cladophora sp.) and terrestrial primary producer (Quercus glauca). Periphyton δ13Cbulk and δ13Cchl values did not necessarily correspond to δ13Cbulk for an algal-grazing specialist (Mayfly larva, Epeorus latifolium), suggesting that periphyton δ13C values do not faithfully trace carbon transfer between primary producers and primary consumers. Periphyton Δ14Cchl values (-258 ‰ in April and -190 ‰ in October) were slightly lower than Δ14Cbulk values (-228 ‰ in April and -179 ‰ in October), but were close to the Δ14C value for dissolved inorganic carbon (DIC) (-217 ± 31 ‰), which is a mixture of weathered carbonates (Δ14C = -1000 ‰) and dissolved atmospheric CO2 (Δ14C approximately +30 ‰ in 2013). Δ14Cchl values were also close to Δ14Cbulk for E. latifolium (-215 ‰ in April and -199 ‰ in October) and Cladophora sp. (-210 ‰), whereas the Δ14Cbulk value for Q. glauca (+27 ‰) was closer to Δ14C for atmospheric CO2. Although the bulk isotopic composition of periphyton is recognised as a surrogate for the photosynthetic algal community, natural periphyton is a mixture of aquatic and terrestrial organic materials. Our results indicate that the bulk periphyton matrix at the study site consists of 89 to 95 % algal carbon (derived from 14C-depleted DIC) and 5 to 11 % terrestrial organic carbon (derived from 14C-enriched atmospheric CO2).

  2. A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling.

    PubMed

    Guan, Shenheng; Price, John C; Prusiner, Stanley B; Ghaemmaghami, Sina; Burlingame, Alma L

    2011-12-01

    In a recent study, in vivo metabolic labeling using (15)N traced the rate of label incorporation among more than 1700 proteins simultaneously and enabled the determination of individual protein turnover rate constants over a dynamic range of three orders of magnitude (Price, J. C., Guan, S., Burlingame, A., Prusiner, S. B., and Ghaemmaghami, S. (2010) Analysis of proteome dynamics in the mouse brain. Proc. Natl. Acad. Sci. U. S. A. 107, 14508-14513). These studies of protein dynamics provide a deeper understanding of healthy development and well-being of complex organisms, as well as the possible causes and progression of disease. In addition to a fully labeled food source and appropriate mass spectrometry platform, an essential and enabling component of such large scale investigations is a robust data processing and analysis pipeline, which is capable of the reduction of large sets of liquid chromatography tandem MS raw data files into the desired protein turnover rate constants. The data processing pipeline described in this contribution is comprised of a suite of software modules required for the workflow that fulfills such requirements. This software platform includes established software tools such as a mass spectrometry database search engine together with several additional, novel data processing modules specifically developed for (15)N metabolic labeling. These fulfill the following functions: (1) cross-extraction of (15)N-containing ion intensities from raw data files at varying biosynthetic incorporation times, (2) computation of peptide (15)N isotopic incorporation distributions, and (3) aggregation of relative isotope abundance curves for multiple peptides into single protein curves. In addition, processing parameter optimization and noise reduction procedures were found to be necessary in the processing modules in order to reduce propagation of errors in the long chain of the processing steps of the entire workflow.

  3. Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O.

    PubMed

    Su, Xun-Cheng; Loh, Choy-Theng; Qi, Ruhu; Otting, Gottfried

    2011-05-01

    Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that reduction of E. coli S30 extracts with NaBH(4) presents a simple and inexpensive way to achieve cleaner selective isotope labelling in cell-free protein synthesis reactions. The purpose of the NaBH(4) is to inactivate all pyridoxal-phosphate (PLP) dependent enzymes by irreversible reduction of the Schiff bases formed between PLP and lysine side chains of the enzymes or amino groups of free amino acids. The reduced S30 extracts retain their activity of protein synthesis, can be stored as well as conventional S30 extracts and effectively suppress conversions between different amino acids. In addition, inactivation of PLP-dependent enzymes greatly stabilizes hydrogens bound to α-carbons against exchange with water, minimizing the loss of α-deuterons during cell-free production of proteins from perdeuterated amino acids in H(2)O solution. This allows the production of highly perdeuterated proteins that contain protons at all exchangeable positions, without having to back-exchange labile deuterons for protons as required for proteins that have been synthesized in D(2)O.

  4. Measurement of (15)N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography-tandem quadrupole mass spectrometry.

    PubMed

    Nakamura, Hidehiro; Karakawa, Sachise; Watanabe, Akiko; Kawamata, Yasuko; Kuwahara, Tomomi; Shimbo, Kazutaka; Sakai, Ryosei

    2015-05-01

    6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional (15)N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M+1 isotopomers of each natural AQC-amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M+1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of (15)N enrichment was confirmed by measuring a series of (15)N-labeled arginine standards. The positional (15)N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional (15)N enrichment in the plasma of rats fed (15)N-labeled glutamine. These results suggest the utility of LC-MS/MS detection of AQC-amino acids for the measurement of isotopic enrichment in (15)N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.

  5. Investigation of Uña De Gato I. 7-Deoxyloganic acid and 15N NMR spectroscopic studies on pentacyclic oxindole alkaloids from Uncaria tomentosa.

    PubMed

    Muhammad, I; Dunbar, D C; Khan, R A; Ganzera, M; Khan, I A

    2001-07-01

    The C-8-(S) isomer of deoxyloganic acid (7-deoxyloganic acid), together with beta-sitosteryl glucoside, five known stereoisomeric pentacyclic oxindole alkaloids and the tetracyclic oxindole isorhyncophylline, were isolated from the inner bark of Uncaria tomentosa. Structures of the isolated compounds were based on 1H and 13C NMR data, mainly 2D NMR experiments, including 1H-13C HMBC and 1H-1H NOESY correlation. Furthermore, the hitherto unreported 15N chemical shifts of the isomeric oxindole alkaloids, using 1H-15N HMBC experiments, were utilized to facilitate their characterization. Uncarine D showed weak cytotoxic activity against SK-MEL, KB, BT-549 and SK-OV-3 cell lines with IC(50) values between 30 and 40 microg/ml, while uncarine C exhibited weak cytotoxicity only against ovarian carcinoma (IC(50) at 37 microg/ml).

  6. High-throughput backbone resonance assignment of small 13C, 15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent 1J ( 1H, 13C): HNCACB codedHAHB

    NASA Astrophysics Data System (ADS)

    Pegan, Scott; Kwiatkowski, Witek; Choe, Senyon; Riek, Roland

    2003-12-01

    The proposed three-dimensional triple-resonance experiment HNCACB codedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13C α, 13C β, 1H α, and 1H β. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1H α and 1H β chemical shifts are then coded in the line shape of the cross-peaks of 13C α, 13C β along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACB codedHAHB experiment was applied to ˜85% labeled 13C, 15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1-77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.

  7. Different fates of deposited NH4+ and NO3- in a temperate forest in northeast China: a (15) N tracer study.

    PubMed

    Liu, Jun; Peng, Bo; Xia, Zongwei; Sun, Jianfei; Gao, Decai; Dai, Weiwei; Jiang, Ping; Bai, Edith

    2017-06-01

    Increasing atmospheric reactive nitrogen (N) deposition due to human activities could change N cycling in terrestrial ecosystems. However, the differences between the fates of deposited NH4+ and NO3- are still not fully understood. Here, we investigated the fates of deposited NH4+ and NO3-, respectively, via the application of (15) NH4 NO3 and NH4(15) NO3 in a temperate forest ecosystem. Results showed that at 410 days after tracer application, most 15NH4+ was immobilized in litter layer (50 ± 2%), while a considerable amount of 15NO3- penetrated into 0-5 cm mineral soil (42 ± 2%), indicating that litter layer and 0-5 cm mineral soil were the major N sinks of NH4+ and NO3-, respectively. Broad-leaved trees assimilated more (15) N under NH4(15) NO3 treatment compared to under (15) NH4 NO3 treatment, indicating their preference for NO3--N. At 410 days after tracer application, 16 ± 4% added (15) N was found in aboveground biomass under 15NO3- treatment, which was twice more than that under 15NH4+ treatment (6 ± 1%). At the same time, approximately 80% added (15) N was recovered in soil and plants under both treatments, which suggested that this forest had high potential for retention of deposited N. These results provided evidence that there were great differences between the fates of deposited NH4+ and NO3-, which could help us better understand the mechanisms and capability of forest ecosystems as a sink of reactive nitrogen. © 2016 John Wiley & Sons Ltd.

  8. Oligomeric complexes of some heteroaromatic ligands and aromatic diamines with rhodium and molybdenum tetracarboxylates: 13C and 15N CPMAS NMR and density functional theory studies.

    PubMed

    Leniak, Arkadiusz; Kamieński, Bohdan; Jaźwiński, Jarosław

    2015-05-01

    Seven new oligomeric complexes of 4,4'-bipyridine; 3,3'-bipyridine; benzene-1,4-diamine; benzene-1,3-diamine; benzene-1,2-diamine; and benzidine with rhodium tetraacetate, as well as 4,4'-bipyridine with molybdenum tetraacetate, have been obtained and investigated by elemental analysis and solid-state nuclear magnetic resonance spectroscopy, (13)C and (15)N CPMAS NMR. The known complexes of pyrazine with rhodium tetrabenzoate, benzoquinone with rhodium tetrapivalate, 4,4'-bipyridine with molybdenum tetrakistrifluoroacetate and the 1 : 1 complex of 2,2'-bipyridine with rhodium tetraacetate exhibiting axial-equatorial ligation mode have been obtained as well for comparison purposes. Elemental analysis revealed 1 : 1 complex stoichiometry of all complexes. The (15)N CPMAS NMR spectra of all new complexes consist of one narrow signal, indicating regular uniform structures. Benzidine forms a heterogeneous material, probably containing linear oligomers and products of further reactions. The complexes were characterized by the parameter complexation shift Δδ (Δδ = δcomplex  - δligand). This parameter ranged from around -40 to -90 ppm in the case of heteroaromatic ligands, from around -12 to -22 ppm for diamines and from -16 to -31 ppm for the complexes of molybdenum tetracarboxylates with 4,4'-bipyridine. The experimental results have been supported by a density functional theory computation of (15)N NMR chemical shifts and complexation shifts at the non-relativistic Becke, three-parameter, Perdew-Wang 91/[6-311++G(2d,p), Stuttgart] and GGA-PBE/QZ4P levels of theory and at the relativistic scalar and spin-orbit zeroth order regular approximation/GGA-PBE/QZ4P level of theory. Nucleus-independent chemical shifts have been calculated for the selected compounds. Copyright © 2015 John Wiley & Sons, Ltd.

  9. Intra-trophic isotopic discrimination of (15)N/(14)N for amino acids in autotrophs: Implications for nitrogen dynamics in ecological studies.

    PubMed

    Takizawa, Yuko; Dharampal, Prarthana S; Steffan, Shawn A; Takano, Yoshinori; Ohkouchi, Naohiko; Chikaraishi, Yoshito

    2017-05-01

    The differential discrimination of nitrogen isotopes ((15)N/(14)N) within amino acids in consumers and their diets has been routinely used to estimate organismal tropic position (TP). Analogous isotopic discrimination can occur within plants, particularly in organs lacking chloroplasts. Such discrimination likely arises from the catabolic deamination of amino acids, resulting in a numerical elevation of estimated TP, within newly synthesized biomass. To investigate this phenomenon, we examined the (15)N/(14)N of amino acids (δ(15) NAA) in spring leaves and flowers from eight deciduous and two annual plants. These plants were classified on the basis of their time of bloom, plants that bloomed when their leaves were absent (Type I) versus plants that bloomed while leaves were already present (Type II). Based on the δ(15) NAA values from leaves, both plant types occupied comparable and ecologically realistic mean TPs (=1.0 ± 0.1, mean ± 1σ). However, the estimated TPs of flowers varied significantly (Type I: 2.2 ± 0.2; Type II: 1.0 ± 0.1). We hypothesize that these results can be interpreted by the following sequence of events: (1) Type I floral biomass is synthesized in absence of active photosynthesis; (2) the catabolic deamination of amino acids in particular, leaves behind (15)N in the residual pool of amino acids; and (3) the incorporation of these (15)N-enriched amino acids within the biomass of Type I flowers results in the numerical elevation of the TPs. In contrast, the actively photosynthesizing Type II leaves energetically sustain the synthesis of Type II flower biomass, precluding any reliance on catabolic deamination of amino acids. Amino acids within Type II flowers are therefore isotopically comparable to the Type II leaves. These findings demonstrate the idiosyncratic nature of the δ(15) NAA values within autotrophic organs and have implications for interpreting trophic hierarchies using primary producers and their consumers.

  10. Solid-State 15N NMR of 15N-Labeled Nylon 6 and Nylon 11

    DTIC Science & Technology

    1990-05-22

    S. Veeman, E. M. Menger, W. Ritchey, and E. de Boer, Macromolecules, 1979, 12, 924. 2. A. N. Garroway , W. M. Ritchey and W. B. Moniz, Macromolecules...S. Veeman and E. M. Menger, Bull. Magn. Reson., 1980, 2, 77. 26. D. L. VanderHart and A. N. Garroway , J. Chem. Phys., 1979, 71, 2773. 27. M. D

  11. Limitations in detection of 15N incorporation by mass spectrometry in protein-based stable isotope probing (protein-SIP).

    PubMed

    Taubert, Martin; von Bergen, Martin; Seifert, Jana

    2013-05-01

    The method of protein-based stable isotope probing (protein-SIP) has previously been shown to allow the modeling of carbon fluxes in microbial communities, thus tackling one of the key questions in microbial ecology. The method allows the analysis of stable isotope distribution in peptides, revealing metabolic activities of the species present in an ecosystem. Besides carbon, an application of protein-SIP with nitrogen is of interest for resolving the nitrogen fluxes in microbial communities. Thus, the sensitivity and reliability of a protein-SIP approach employing (15)N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of (14)N/(15)N were performed, from 10 % down to 0.1 % (15)N. After incubation leading to complete labeling of biomass, proteins were extracted and separated by one-dimensional gel electrophoresis, followed by tryptic digest and UPLC Orbitrap MS/MS analysis. (15)N relative isotope abundance (RIA) was calculated based on isotopic patterns from identified peptides in mass spectra. Proteomics data have been deposited to ProteomeXchange with identifier PXD000127. The distribution of (15)N RIA values among peptides was analyzed in samples with different (15)N amount, and potential causes for variations within individual samples of either technical or biological origin were investigated. Using a number of 50 peptides, significant differences (p ≤ 0.05) in (15)N incorporation were found between samples of different (15)N RIA down to 0.1 %. The study demonstrates that protein-SIP using (15)N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.

  12. Small Cab-like proteins retard degradation of photosystem II-associated chlorophyll in Synechocystis sp. PCC 6803: kinetic analysis of pigment labeling with 15N and 13C.

    PubMed

    Vavilin, Dmitrii; Yao, Danny; Vermaas, Wim

    2007-12-28

    Isotope (Na(15)NO(3), ((15)NH(4))SO(4) or [(13)C]glucose) labeling was used to analyze chlorophyll synthesis and degradation rates in a set of Synechocystis mutants that lacked single or multiple small Cab-like proteins (SCPs), as well as photosystem I or II. When all five small Cab-like proteins were inactivated in the wild-type background, chlorophyll stability was not affected unless the scpABCDE(-) strain was grown at a moderately high light intensity of 100-300 micromol photons m(-2) s(-1). However, the half-life time of chlorophyll was 5-fold shorter in the photosystem I-less/scpABCDE(-) strain than in the photosystem I-less strain even when grown at low light intensity (~3 micromol photons m(-2) s(-1)) (32 +/- 5 and 161 +/- 25 h, respectively). In other photosystem I-less mutants that lacked one to four of the scp genes the chlorophyll lifetime was in between these two values, with the chlorophyll lifetime generally decreasing with an increasing number of inactivated scps. In contrast, the chlorophyll biosynthesis rate was only marginally affected by inactivation of scps except when all five scp genes were deleted. Small Cab-like protein deficiency did not significantly affect photoinhibition or turnover of photosystem II-associated beta-carotene. It is concluded that SCPs do not alter the stability of functional photosystem II complexes but retard the degradation of photosystem II-associated chlorophyll, consistent with the proposed involvement of SCPs in photosystem II re-assembly or/and repair processes by temporarily binding chlorophyll while photosystem II protein components are being replaced.

  13. NMR studies of two spliced leader RNAs using isotope labeling

    SciTech Connect

    Lapham, J.; Crothers, D.M.

    1994-12-01

    Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions between the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.

  14. New insight in tholin chemical structure through 13C and 15N solid state nuclear magnetic resonance

    NASA Astrophysics Data System (ADS)

    Derenne, S.; Coelho, C.; Anquetil, C.; Szopa, C.; Quirico, E.; Bonhomme, C.

    2011-10-01

    Tholins are complex materials synthesized in laboratory as models of the molecules occurring in the atmosphere of Titan. Using labeled gases, pure 13C and 15N-enriched tholins were synthesized and analyzed using 13C and 15N nuclear magnetic resonance. This study allowed confirming the presence of some functional groups (cyano, amino, imino) previously inferred from other techniques and to assess their relative contribution. It also indicated that some other functions (such as carbodiimide, protonated aromatic carbons) if present, only show a very low contribution and ruled out the occurrence of hydrazones.

  15. Nitrogen cycling in a forest stream determined by a 15N tracer addition

    Treesearch

    Patrick J. Mullholland; Jennifer L. Tank; Diane M. Sanzone; Wilfred M. Wollheim; Bruce J. Peterson; Jackson R. Webster; Judy L. Meyer

    2000-01-01

    Nitrogen uptake and cycling was examined using a six-week tracer addition of 15N-labeled ammonium in early spring in Waer Branch, a first-order deciduous forest stream in eastern Tennessee. Prior to the 15N addition, standing stocks of N were determined for the major biomass compartments. During and after the addition,

  16. 15N, 17O NMR and X-ray diffraction study of mesoionic 1,2,3,4-thiatriazolium-5-olate and its ethylated derivative

    NASA Astrophysics Data System (ADS)

    Jaźiwińsk, J.; Staszewska, O.; Staszewski, P.; Stefaniak, L.; Wiench, J. W.; Webb, G. A.

    1999-02-01

    Two mesoionic compounds with oxygenous exocyclic groups: 3-phenyl-1,2,3,4-thiatriazolium-5-olate 1 and its ethylated derivative 2 were investigated by means of 15N, 17O NMR and X-ray diffraction techniques. The exocyclic C5-O6 bond of thiatriazole 1 [1.224(3) Å] has a strong double bond character. Bond lengths in the thiatriazole ring are intermediate between single and double bond values except for S1-C5 [1.800(2) Å] which is close to a single Csp 3-S bond. The C5-O6 bond is significantly longer for the ethylated derivative 2 [1.314(4) Å]. The ethyl group attached to O6 is located in the trans position in relation to the ring S1 atom. The experimental data are compared with the results of ab initio molecular orbital calculations. The calculated absolute nuclear shieldings, chemical shifts and charge densities, in spite of some limitations, can be useful as an aid to signal assignments and for an understanding of the NMR parameters.

  17. [1H, 15N] heteronuclear single quantum coherence NMR study of the mechanism of aquation of platinum(IV) ammine complexes.

    PubMed

    Davies, Murray S; Hall, Matthew D; Berners-Price, Susan J; Hambley, Trevor W

    2008-09-01

    The aquation and hydrolysis of a series of platinum(IV) complexes of the general form cis, trans, cis-[PtCl 2(X) 2( (15)NH 3) 2] (X = Cl (-), O 2CCH 3 (-), OH (-)) have been followed by [ (1)H, (15)N] Heteronuclear Single Quantum Coherence NMR spectroscopy. Negligible aquation (<5%) is observed for the complexes where X = O 2CCH 3 (-) or OH (-) over 3-4 weeks. Aquation of cis-[PtCl 4( (15)NH 3) 2] ( 1) is observed, and the rate of aquation increases with increasing pH and upon the addition of 0.01 mol equiv of the platinum(II) complex cis-[PtCl 2( (15)NH 3) 2] (cisplatin). The first aquated species formed from cis-[PtCl 4(NH 3) 2] has one of the axial chloro groups (relative to the equatorial NH 3 ligands) replaced by an aqua/hydroxo ligand. The second observed substitution occurs in an equatorial position. Peaks that are consistent with five of the eight possible aquation species were observed in the NMR spectra.

  18. NMR studies of the POU-specific DNA-binding domain of Oct-1: sequential 1H and 15N assignments and secondary structure.

    PubMed

    Cox, M; Dekker, N; Boelens, R; Verrijzer, C P; van der Vliet, P C; Kaptein, R

    1993-06-15

    The 1H and 15N resonances of the POU-specific DNA-binding domain of transcription factor Oct-1 have been assigned sequentially using two-dimensional homo- and heteronuclear NMR techniques, as well as three-dimensional heteronuclear NMR techniques, including TOCSY, 2D NOE, and NOESY-HMQC experiments. A number of typical short- and medium-range NOE contacts, as well as amide proton exchange data, gave evidence for the presence of four alpha-helices, in the peptide segments 1-19, 23-34, 40-49, and 54-71, which are connected by short loops of irregular structure. Interestingly, the second helix contains three glycine residues and the fourth helix a proline in the middle of the helix. Although the regular pattern of hydrogen bonds in the fourth helix is interrupted, due to the absence of an amide proton in proline, the helix is remarkably stable. All four helices are amphipathic, which suggests a packing of the apolar sides of the helices in the folded structure of the protein.

  19. Nitrogen input 15N-signatures are reflected in plant 15N natural abundances of N-rich tropical forest in China

    NASA Astrophysics Data System (ADS)

    Abdisa Gurmesa, Geshere; Lu, Xiankai; Gundersen, Per; Yunting, Fang; Mo, Jiangming

    2016-04-01

    In this study, we tested the measurement of natural abundance of 15N15N) for its ability to assess changes in N cycling due to increased N deposition in two forest types; namely, an old-growth broadleaved forest and a pine forest, in southern China. We measured δ15N values of inorganic N in input and output fluxes under ambient N deposition, and N concentration and δ15N of major ecosystem compartments under ambient and increased N deposition. Our results showed that N deposition to the forests was 15N-depleted, and was dominated by NH4-N. Plants were 15N-depleted due to imprint from the 15N-depleted atmospheric N deposition. The old-growth forest had larger N concentration and was more 15N-enriched than the pine forest. Nitrogen addition did not significantly affect N concentration, but it significantly increased δ15N values of plants, and slightly more so in the pine forest, toward the 15N signature of the added N in both forests. The result indicates that the pine forest may rely more on the 15N-depleted deposition N. Soil δ15N values were slightly decreased by the N addition. Our result suggests that ecosystem δ15N is more sensitive to the changes in ecosystem N status and N cycling than N concentration in N-saturated sub-tropical forests.

  20. Quantitative role of splanchnic region in leucine metabolism: L-(1-13C,15N)leucine and substrate balance studies

    SciTech Connect

    Yu, Y.M.; Wagner, D.A.; Tredget, E.E.; Walaszewski, J.A.; Burke, J.F.; Young, V.R. )

    1990-07-01

    The role of the splanchnic region (Sp) in whole body leucine metabolism was assessed in six chronically catheterized fasting mongrel dogs and in eight dogs during constant enteral feeding of a complete amino acid solution (0.24 g.kg-1.h-1). We used primed continuous intravenous infusions of L-(1-13C,15N)leucine and L-(1-14C)leucine and measurements of arteriovenous isotope and leucine balance across the gut, liver, and Sp. In the fasted condition, 3.5% of arterial leucine supply was oxidized in the Sp, accounting for 13% of total body leucine oxidation, with 10% by liver. With amino acid feeding (1) leucine carbon and nitrogen fluxes and oxidation were increased (P less than 0.01) at the whole body level; (2) the percent of whole body leucine oxidation occurring in the Sp and liver increased (P less than 0.01) to 41 and 27%, respectively; (3) fractional metabolic utilization of leucine delivered to the Sp was reduced (P less than 0.01) from 47 to 35%; (4) the deamination rate of leucine in the gut was increased (P less than 0.05), along with an increased reamination rate of alpha-ketoisocaproic acid in the Sp (P less than 0.05). These findings reveal that the Sp accounts for a small fraction of whole body leucine oxidation during the fasting condition, but it plays a quantitatively important role in total body leucine oxidation during amino acid feeding; the gut and liver play cooperative roles in controlling leucine supply to peripheral tissues.

  1. Revisiting the use of δ15N in meso-scale studies of marine food webs by considering spatio-temporal variations in stable isotopic signatures - The case of an open ecosystem: The Bay of Biscay (North-East Atlantic)

    NASA Astrophysics Data System (ADS)

    Chouvelon, T.; Spitz, J.; Caurant, F.; Mèndez-Fernandez, P.; Chappuis, A.; Laugier, F.; Le Goff, E.; Bustamante, P.

    2012-08-01

    Most of the recent framework directives and environmental policies argue for the development and the use of indicators - notably trophodynamic indicators - that should be able to follow ecosystems' evolution in space and time, particularly under anthropogenic perturbations. In the last decades, the use of stable carbon and nitrogen isotopes ratios has increased exponentially, particularly in studies of marine ecosystems' trophic structure and functioning. This method is principally based on the assumption that the isotopic composition of a consumer directly reflects that of its food. Nevertheless, few studies have attempted to define the limits of this tool, before using it and drawing ecological conclusions from isotopic analysis. This study aimed to assess the importance of considering spatio-temporal variations in isotopic signatures of consumers when using δ13C and especially δ15N values in open ecosystems with complex food webs, using the Bay of Biscay (North-East Atlantic) as a case study. To this end, more than 140 species from this marine ecosystem were analysed for the isotopic signatures in their muscle tissue. They were sampled from coastal to oceanic and deep-sea areas and at different latitudes, to evaluate spatial variations of isotopic signatures. Selected species were also sampled over several years and in two seasons to account for inter-annual and seasonal variations. In the Bay of Biscay temperate ecosystem, which is subject to both coastal and oceanic influences - two main river inputs and upwelling areas - , δ13C and δ15N values significantly decreased from inshore to offshore species, and to a lesser extent from benthic to pelagic organisms. River discharges appeared to be the first factor influencing δ13C and δ15N values in consumers. From the important spatial variations detected in δ15N values in particular, we suggest that in such contrasted ecosystem, nitrogen isotopic ratios may also be revisited as an indicator of the feeding

  2. Application of (13)C and (15)N stable isotope probing to characterize RDX degrading microbial communities under different electron-accepting conditions.

    PubMed

    Cho, Kun-Ching; Lee, Do Gyun; Fuller, Mark E; Hatzinger, Paul B; Condee, Charles W; Chu, Kung-Hui

    2015-10-30

    This study identified microorganisms capable of using the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) or its metabolites as carbon and/or nitrogen sources under different electron-accepting conditions using (13)C and (15)N stable isotope probing (SIP). Mesocosms were constructed using groundwater and aquifer solids from an RDX-contaminated aquifer. The mesocosms received succinate as a carbon source and one of four electron acceptors (nitrate, manganese(IV), iron(III), or sulfate) or no additional electron acceptor (to stimulate methanogenesis). When RDX degradation was observed, subsamples from each mesocosm were removed and amended with (13)C3- or ring-(15)N3-, nitro-(15)N3-, or fully-labeled (15)N6-RDX, followed by additional incubation and isolation of labeled nucleic acids. A total of fifteen 16S rRNA sequences, clustering in α- and γ-Proteobacteria, Clostridia, and Actinobacteria, were detected in the (13)C-DNA fractions. A total of twenty seven sequences were derived from different (15)N-DNA fractions, with the sequences clustered in α- and γ-Proteobacteria, and Clostridia. Interestingly, sequences identified as Desulfosporosinus sp. (in the Clostridia) were not only observed to incorporate the labeled (13)C or (15)N from labeled RDX, but also were detected under each of the different electron-accepting conditions. The data suggest that (13)C- and (15)N-SIP can be used to characterize microbial communities involved in RDX biodegradation, and that the dominant pathway of RDX biodegradation may differ under different electron-accepting conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Phenylalanine δ15N in Paleo Archives as a New Proxy for δ15N of Exported Primary Production

    NASA Astrophysics Data System (ADS)

    McCarthy, M.; Batista, F. C.; Vokhshoori, N. L.; Brown, J. T.; Guilderson, T. P.; Ravelo, A. C.; Sherwood, O.

    2012-12-01

    Compound-specific isotope analysis of individual amino acids (CSI-AA) is emerging as a powerful new tool for studying the paleo nitrogen cycle. Because most detrital organic nitrogen is composed of amino acids, CSI-AA can reveal the mechanistic basis for organic nitrogen diagenesis, preserve a record of past food web structure, and potentially reconstruct the δ15N values of past nitrate and primary production. Within the commonly measured amino acids, the δ15N value of phenylalanine (Phe) appears uniquely promising as a new proxy that reflects the nitrogen isotopic value of the original source. Phe δ15N values remain almost unchanged with trophic transfer through food webs, and also during at least the initial stages of organic matter degradation. Here we synthesize results from both bio-archives and recent sediments, which together suggest that at least in Holocene archives the Phe δ15N value does in fact record the average inorganic nitrogen δ15N value at the base of planktonic food webs. However, several important unknowns also remain. These include the extent of variation in amino acid isotopic fractionation patterns in phylogenetically distinct algal groups. The stability of Phe δ15N values in older sediments where organic matter has undergone extensive diagenesis is also an important research area, which may ultimately establish the temporal limit for application of this approach to study past geological epochs. Together, however, results to date suggest that of Phe δ15N values in paleo archives represent a novel molecular-level proxy which is not tied to any specific organism or group, but rather can provide an integrated estimate of δ15N value of exported primary production.

  4. Plan of study to determine if the isotopic ratios [delta]15 N and [delta]18 O can reveal the sources of nitrate discharged by the Mississippi River into the Gulf of Mexico

    USGS Publications Warehouse

    Battaglin, William A.; Kendall, Carol; Goolsby, Donald A.; Boyer, Laurie L.

    1997-01-01

    Nitrate and other nutrients discharged from the Mississippi River basin are suspected of causing a zone of depleted dissolved oxygen (hypoxic zone) in the Gulf of Mexico each summer. The hypoxic zone may have an adverse effect on aquatic life and commercial fisheries. Commercial fertilizers are the dominant source of nitrogen input to the Mississippi basin. Other nitrogen sources include animal waste, fixation of atmospheric nitrogen by legumes, precipitation, domestic and industrial effluent, and the soil. The inputs of nitrogen from most of these sources to the Mississippi basin can be estimated and the outputs in surface water can be measured. However, nitrogen from each source is affected differently by physical, chemical, and biological processes that control nitrogen cycling in terrestrial and aquatic systems. Hence, the relative contributions from the various sources of nitrogen to nitrate load in the Mississippi River are unknown because the different sources may not contribute proportionally to their inputs in the basin. It may be possible to determine the relative contributions of the major sources of nitrate in river water using the stable isotopic ratios d15N and d18O of the nitrate ion. A few researchers have used the d15N and/or d18O isotope ratios to determine sources of nitrate in ground water, headwater catchments, and small rivers, but little is known about the isotopic composition of nitrate in larger rivers. The objective of this study is to measure the isotopic composition of nitrate and suspended organic matter in the Mississippi River and its major tributaries, in discharge to the Gulf of Mexico, and in streamflow from smaller watersheds that have distinct sources of nitrogen (row crops, animal wastes, and urban effluents) or are minimally impacted by man (undeveloped). Samples from seven sites on the Mississippi River and its tributaries and from 17 sites in smaller watersheds within the Mississippi River basin will be analyzed for d15N and

  5. Probing site-specific 13C/15N-isotope enrichment of spider silk with liquid-state NMR spectroscopy.

    PubMed

    Shi, Xiangyan; Yarger, Jeffery L; Holland, Gregory P

    2013-05-01

    Solid-state nuclear magnetic resonance (NMR) has been extensively used to elucidate spider silk protein structure and dynamics. In many of these studies, site-specific isotope enrichment is critical for designing particular NMR methods for silk structure determination. The commonly used isotope analysis techniques, isotope-ratio mass spectroscopy and liquid/gas chromatography-mass spectroscopy, are typically not capable of providing the site-specific isotope information for many systems because an appropriate sample derivatization method is not available. In contrast, NMR does not require any sample derivatization or separation prior to analysis. In this article, conventional liquid-state (1)H NMR was implemented to evaluate incorporation of (13)C/(15)N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific (13)C and (15)N isotope enrichments, an analysis method was developed to fit the (1)H-(13)C and (1)H-(15)N J-splitting (J CH and J NH) (1)H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U-(13)C3,(15)N]-Ala and [1-(13)C,(15)N]-Gly were dissolved in their water supplies. Overall, contents for Ala and Gly isotopomers are extracted for these silk samples. The current methodology can be applied to many fields where site-specific tracking of isotopes is of interest.

  6. Solvent effects on 15N NMR coordination shifts.

    PubMed

    Kleinmaier, Roland; Arenz, Sven; Karim, Alavi; Carlsson, Anna-Carin C; Erdélyi, Máté

    2013-01-01

    (15)N NMR chemical shift became a broadly utilized tool for characterization of complex structures and comparison of their properties. Despite the lack of systematic studies, the influence of solvent on the nitrogen coordination shift, Δ(15)N(coord), was hitherto claimed to be negligible. Herein, we report the dramatic impact of the local environment and in particular that of the interplay between solvent and substituents on Δ(15)N(coord). The comparative study of CDCl(3) and CD(3)CN solutions of silver(I)-bis(pyridine) and silver(I)-bis(pyridylethynyl)benzene complexes revealed the strong solvent dependence of their (15)N NMR chemical shift, with a solvent dependent variation of up to 40 ppm for one and the same complex. The primary influence of the effect of substituent and counter ion on the (15)N NMR chemical shifts is rationalized by corroborating Density-Functional Theory (nor discrete Fourier transform) calculations on the B3LYP/6-311 + G(2d,p)//B3LYP/6-31G(d) level. Cooperative effects have to be taken into account for a comprehensive description of the coordination shift and thus the structure of silver complexes in solution. Our results demonstrate that interpretation of Δ(15)N(coord) in terms of coordination strength must always consider the solvent and counter ion. The comparable magnitude of Δ(15)N(coord) for reported transition metal complexes makes the principal findings most likely general for a broad scale of complexes of nitrogen donor ligands, which are in frequent use in modern organometallic chemistry.

  7. Solid-phase peptide synthesis and solid-state NMR spectroscopy of (Ala/sup 3/-/sup 15/N)(Val/sup 1/)gramicidin A

    SciTech Connect

    Fields, G.B.; Fields, C.G.; Petefish, J.; Van Wart, H.E.; Cross, T.A.

    1988-03-01

    (Ala/sup 3-15/N)(Val/sup 1/)Gramicidin A has been prepared by solid-phase peptide synthesis and studied by solid-state /sup 15/N nuclear magnetic resonance spectroscopy. The synthesis of desformyl(Ala/sup 3-15/N)(Val/sup 1/)gramicidin A employed N-hydroxysuccinimide esters of 9-fluorenylmethoxycarbonyl-N/sup ..cap alpha../-amino acids and completely avoided the use of acid. Since deblocking was done with piperidine and the peptide was removed from the resin by treatment with ethanolamine, this synthetic protocol prevented oxidation of the indole rings of this tryptophan-rich peptide and reduced truncations produced by acid hydrolysis. After formylation and purification by anion-exchange and high-pressure liquid chromatography, the peptide was obtained in an overall yield of 30%. Solid-state /sup 15/N nuclear magnetic resonance spectra of this peptide and uniformly labeled (/sup 15/N)gramicidin A' oriented in hydrated lipid bilayers have been obtained, allowing unambiguous assignment of the (/sup 15/N)Ala/sup 3/ resonance in the latter. The solid-state /sup 15/N nuclear magnetic resonance experiments provide evidence that (Val/sup 1/)gramicidin A is rotating about an axis that is perpendicular to the plane of the lipid bilayer and that the N-H axis is nearly parallel with the rotational axis. This study demonstrates that site-specifically labeled (/sup 15/N)gramicidin A analogs prepared by solid-phase peptide synthesis are valuable tools in the study of the solid-state nuclear magnetic resonance spectra of samples in oriented lipid bilayers.

  8. 15N Hyperpolarization by Reversible Exchange Using SABRE-SHEATH

    PubMed Central

    2016-01-01

    NMR signal amplification by reversible exchange (SABRE) is a NMR hyperpolarization technique that enables nuclear spin polarization enhancement of molecules via concurrent chemical exchange of a target substrate and parahydrogen (the source of spin order) on an iridium catalyst. Recently, we demonstrated that conducting SABRE in microtesla fields provided by a magnetic shield enables up to 10% 15N-polarization (Theis, T.; et al. J. Am. Chem. Soc.2015, 137, 1404). Hyperpolarization on 15N (and heteronuclei in general) may be advantageous because of the long-lived nature of the hyperpolarization on 15N relative to the short-lived hyperpolarization of protons conventionally hyperpolarized by SABRE, in addition to wider chemical shift dispersion and absence of background signal. Here we show that these unprecedented polarization levels enable 15N magnetic resonance imaging. We also present a theoretical model for the hyperpolarization transfer to heteronuclei, and detail key parameters that should be optimized for efficient 15N-hyperpolarization. The effects of parahydrogen pressure, flow rate, sample temperature, catalyst-to-substrate ratio, relaxation time (T1), and reversible oxygen quenching are studied on a test system of 15N-pyridine in methanol-d4. Moreover, we demonstrate the first proof-of-principle 13C-hyperpolarization using this method. This simple hyperpolarization scheme only requires access to parahydrogen and a magnetic shield, and it provides large enough signal gains to enable one of the first 15N images (2 × 2 mm2 resolution). Importantly, this method enables hyperpolarization of molecular sites with NMR T1 relaxation times suitable for biomedical imaging and spectroscopy. PMID:25960823

  9. Use of 15N labeled Urea to Study Nitrogen Use Efficiencies in a Forage Corn-Oat Rotation Grown on Clay and Sandy Clay Loam Soils of Mexico

    USDA-ARS?s Scientific Manuscript database

    Nitrogen (N) is applied in large amounts to sustain high forage yield and quality to feed a dairy cattle population surpassing 440,000 heads in Comarca Lagunera, Mexico (25° 32´ N, 103° 14´W and 1150 masl). Traditional farm practices across this region overaplly organic (manure) and fertilizer N. We...

  10. Use of specifically {sup 15}N-labeled histidine to study structures and mechanisms within the active sites of serine proteinases

    SciTech Connect

    Bachovchin, W.W.

    1994-12-01

    The current emphasis in biological NMR work is on determining structures of biological macromolecules in solution. This emphasis is appropriate because NMR is the only technique capable of providing high-resolution structures that are comparable to those of x-ray crystallography for molecules in solution. This structural knowledge is immensely valuable and is needed in many areas of investigation. However, as valuable as such structural knowledge is, it never provides all the answers; a structure often reveals more questions than answers.

  11. Leveraging Label-Independent Features for Classification in Sparsely Labeled Networks: An Empirical Study

    NASA Astrophysics Data System (ADS)

    Gallagher, Brian; Eliassi-Rad, Tina

    We address the problem of within-network classification in sparsely labeled networks. Recent work has demonstrated success with statistical relational learning (SRL) and semi-supervised learning (SSL) on such problems. However, both approaches rely on the availability of labeled nodes to infer the values of missing labels. When few labels are available, the performance of these approaches can degrade. In addition, many such approaches are sensitive to the specific set of nodes labeled. So, although average performance may be acceptable, the performance on a specific task may not. We explore a complimentary approach to within-network classification, based on the use of label-independent (LI) features - i.e., features calculated without using the values of class labels. While previous work has made some use of LI features, the effects of these features on classification performance have not been extensively studied. Here, we present an empirical study in order to better understand these effects. Through experiments on several real-world data sets, we show that the use of LI features produces classifiers that are less sensitive to specific label assignments and can lead to performance improvements of over 40% for both SRL- and SSL-based classifiers. We also examine the relative utility of individual LI features; and show that, in many cases, it is a combination of a few diverse network-based structural characteristics that is most informative.

  12. Regional patterns in foliar 15N across a gradient of nitrogen deposition in the northeastern US

    Treesearch

    Linda H. Pardo; Steven G. McNulty; Johnny L. Boggs; Sara Duke

    2007-01-01

    Recent studies have demonstrated that natural abundance 15N can be a useful tool for assessing nitrogen saturation, because as nitrification and nitrate loss increase, a15N of foliage and soil also increases. We measured foliar a15N at 11 high-elevation spruce-fir stands along an N deposition gradient...

  13. Regional patterns in foliar 15N across a gradient of nitrogen deposition in the northeastern US

    Treesearch

    Linda H. Pardo; Steven G. McNulty; Johnny L. Boggs; Sara Duke

    2007-01-01

    Recent studies have demonstrated that natural abundance 15N can be a useful tool for assessing nitrogen saturation, because as nitrification and nitrate loss increase, d15N of foliage and soil also increases. We measured foliar d15N at 11 high-elevation spruce-fir stands along an N deposition gradient...

  14. A novel method for trapping and analyzing 15N in NO for tracing NO sources

    NASA Astrophysics Data System (ADS)

    Kang, Ronghua; Mulder, Jan; Dörsch, Peter

    2016-04-01

    15N isotope tracing is an effective and direct approach to investigate the biological and chemical sources of nitric oxide (NO) in soil. However, NO is highly reactive and rapidly converted to nitrogen dioxide (NO2) in the presence of ozone. Various chemical conversions of NO to the more stable solutes nitrite (NO2-) and nitrate (NO3-) have been proposed, which allow analysing the 15N abundance without major fractionation. However, NO emissions from soils are usually small, posing major challenges to conversion efficiency and background contamination. Here we present a novel method in which NO is oxidized to NO2- by chromium trioxide (CrO3) prior to conversion to NO2- and NO3- in an alkaline hydrogen peroxide (H2O2) solution. Immediately following trapping, manganese dioxide (MnO2) and 5M HCl are added to remove excess H2O2, and to adjust the pH to around 6.0-7.0, respectively. The resulting solution can be stored until analysis and is none-toxic, allowing to use a modified denitrifier method (Zhu et al., submitted), where NO2- and NO3- are reduced quantitatively to nitrous oxide (N2O). Optimum NO conversion rates of > 90% even at extremely low initial NO concentration were obtained with 4% H2O2, 0.5 M NaOH, and 0.5 L min-1 gas flow rate. In a laboratory test, using NO gas with different 15N signals produced from unlabelled and labelled NO2-, we found an overall precision of 0.4‰ for unlabelled and 49.7‰ for NO enriched with 1.0 atom% 15N, respectively. This indicates that this method can be used for both natural abundance studies of NO, as well as in labelling studies tracing NO sources. Zhu J, Yu L, Bakken LR, Mørkved PT, Mulder J, Dörsch P. Controlled induction of denitrification in Pseudomonas aureofaciens: a modified denitrifier method for 15N and 18O analysis in NO3- from natural water samples by IRMS. Submitted.

  15. Selectively labeling the heterologous protein in Escherichia coli for NMR studies: a strategy to speed up NMR spectroscopy.

    PubMed

    Almeida, F C; Amorim, G C; Moreau, V H; Sousa, V O; Creazola, A T; Américo, T A; Pais, A P; Leite, A; Netto, L E; Giordano, R J; Valente, A P

    2001-01-01

    Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.

  16. Potential for assessing long-term dynamics in soil nitrogen availability from variations in delta15N of tree rings.

    PubMed

    Hart, S C; Classen, A T

    2003-03-01

    Numerous researchers have used the isotopic signatures of C, H, and O in tree rings to provide a long-term record of changes in the physiological status, climate, or water-source use of trees. The frequently limiting element N is also found in tree rings, and variation in its isotopic signature may provide insight into long-term changes in soil N availability of a site. However, research has suggested that N is readily translocated among tree ring of different years; such infidelity between the isotopic compositions of the N taken up from the soil and the N contained in the ring of that growth year would obscure the long-term N isotopic record. We used a 15-year 15N-tracer study to assess the degree of N translocation among tree rings in ponderosa pine (Pinus ponderosa) trees growing in a young, mixed-conifer plantation. We also measured delta13C and delta15N values in unlabeled trees to assess the degree of their covariance in wood tissue, and to explore the potential for a biological linkage between them. We found that the maximum delta15N values in rings from the labeled trees occurred in the ring formed one-year after the 15N was applied to the roots. The delta15N value of rings from labeled trees declined exponentially and bidirectionally from this maximum peak, toward younger and older rings. The unlabeled trees showed considerable interannual variation in the delta15N values of their rings (up to 3 and 5 per thousand), but these values correlated poorly between trees over time and differed by as much as 6 per thousand. Removal of extractives from the wood reduced their delta15N value, but the change was fairly small and consistent among unlabeled trees. The delta13C and delta15N values of tree rings were correlated over time in only one of the unlabeled trees. Across all trees, both delta13C values of tree rings and annual stem wood production were well correlated with annual precipitation, suggesting that soil water balance is an important environmental

  17. Simultaneous measurement of 13C- and 15N-isotopic enrichments of threonine by mass spectrometry.

    PubMed

    Godin, Jean-Philippe; Mermoud, Anne-France; Rémond, Didier; Faure, Magali; Breuille, Denis; Williamson, Gary; Peré-Trepat, Emma; Ramadan, Ziad; Fay, Laurent-Bernard; Kochhar, Sunil

    2009-04-01

    Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic enrichments of differently labelled chemical compounds (e.g. (13)C and (15)N in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic enrichments of doubly labelled threonine ([U(13)C] and (15)N) with isotopic enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. To calculate both isotopic enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. Moreover, the accuracy of GC/MS (13)C-isotopic enrichment measured was validated by analyzing the same plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Statistical analysis showed that both techniques gave the same results (ANOVA, P >0.05). This new GC/MS method offers the possibility to measure (13)C- and (15)N-isotopic enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS.

  18. Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.

    PubMed

    Gómez-Cortés, Pilar; Brenna, J Thomas; Sacks, Gavin L

    2012-06-19

    Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve

  19. Solution-state (15)N NMR spectroscopic study of alpha-C-phycocyanin: implications for the structure of the chromophore-binding pocket of the cyanobacterial phytochrome Cph1.

    PubMed

    Hahn, Janina; Kühne, Ronald; Schmieder, Peter

    2007-12-17

    The detailed structure of the chromophore-binding pocket in phytochrome proteins and the structural changes associated with its photocycle are still matters of debate. Insight into the structure and dynamics of the binding pocket has been gained through the comparison of a (15)N NMR spectrum of alpha-C-phycocyanin, which is often used as a model system for the study of phytochromes, with the previously described (15)N NMR spectrum of the cyanobacterial phytochrome Cph1. The former spectrum supports the hypothesis that all four nitrogen atoms of the alpha-C-phycocyanin chromophore are protonated, in analogy with the proposed protonation state for the P(r) and P(fr) forms of Cph1. The spectra show that the chromophores in both proteins exhibit a distinct dynamic behavior, as also indicated by a NOESY spectrum of Cph1. Finally, stereochemical arguments and a Cph1 homology model support the hypothesis that the chromophore in Cph1 is most likely in the ZZZssa conformation in the P(r) form of the protein.

  20. Study of stereospecificity of 1H, 13C, 15N and 77Se shielding constants in the configurational isomers of the selenophene-2-carbaldehyde azine by NMR spectroscopy and MP2-GIAO calculations.

    PubMed

    Afonin, Andrei V; Pavlov, Dmitry V; Albanov, Alexander I; Levanova, Ekaterina P; Levkovskaya, Galina G

    2011-11-01

    In the (1)H and (13)C NMR spectra of selenophene-2-carbaldehyde azine, the (1)H-5, (13)C-3 and (13)C-5 signals of the selenophene ring are shifted to higher frequencies, whereas those of the (1)H-1, (13)C-1, (13)C-2 and (13)C-4 are shifted to lower frequencies on going from the EE to ZZ isomer or from the E moiety to the Z moiety of EZ isomer. The (15)N chemical shift is significantly larger in the EE isomer relative to the ZZ isomer and in the E moiety relative to the Z moiety of EZ isomer. A very pronounced difference (60-65 mg/g) between the (77)Se resonance positions is revealed in the studied azine isomers, the (77)Se peak being shifted to higher frequencies in the ZZ isomer and in the Z moiety of EZ isomer. The trends in the changes of the measured chemical shifts are reasonably reproduced by the GIAO calculations at the MP2 level of the (1)H, (13)C, (15)N and (77)Se shielding constants in the energy-favorable conformation with the syn orientation of both selenophene rings relative to the C = N groups. The NBO analysis suggests that such an arrangement of the selenophene rings may take place because of a higher energy of some intramolecular interactions. Copyright © 2011 John Wiley & Sons, Ltd.

  1. 15N and13C NMR investigation of hydroxylamine-derivatized humic substances

    USGS Publications Warehouse

    Thorn, K.A.; Arterburn, J.B.; Mikita, M.A.

    1992-01-01

    Five fulvic and humic acid samples of diverse origins were derivatized with 15N-labeled hydroxylamine and analyzed by liquid-phase 15N NMR spectrometry. The 15N NMR spectra indicated that hydroxylamine reacted similarly with all samples and could discriminate among carbonyl functional groups. Oximes were the major derivatives; resonances attributable to hydroxamic acids, the reaction products of hydroxylamine with esters, and resonances attributable to the tautomeric equilibrium position between the nitrosophenol and monoxime derivatives of quinones, the first direct spectroscopic evidence for quinones, also were evident. The 15N NMR spectra also suggested the presence of nitriles, oxazoles, oxazolines, isocyanides, amides, and lactams, which may all be explained in terms of Beckmann reactions of the initial oxime derivatives. INEPT and ACOUSTIC 15N NMR spectra provided complementary information on the derivatized samples. 13C NMR spectra of derivatized samples indicated that the ketone/quinone functionality is incompletely derivatized with hydroxylamine. ?? 1991 American Chemical Society.

  2. 15N investigation into the effect of a pollutant on the nitrogen metabolism of Tetrahymena pyriformis as a model for environmental medical research.

    PubMed Central

    Arndt, K; Hofmann, D; Gehre, M; Krumbiegel, P

    1998-01-01

    A pilot study was performed to examine the potential of stable isotope techniques for monitoring the impact of a harmful substance on the cellular nitrogen metabolism in the ciliate species Tetrahymena pyriformis. After identical cultivation periods of control cells and toluene-exposed cells in a defined culture medium enriched with [guanidino-15N2]l-arginine, a number of nitrogen-containing pools were analyzed: 1) quantity and 15N abundance of ammonia as the end product of nitrogen metabolism in the system; 2) pattern and 15N abundances of the protein-bound amino acids in the cells; 3) pattern and 15N abundances of free amino acids in the cells; and 4) pattern and 15N abundances of the amino acids in the culture medium. In addition to 15N emission spectrometry, a new gas chromatography/combustion interface-isotope ratio mass spectrometry/mass spectrometry analytical system was used. The production and 15N content of ammonia were higher in the toluene-exposed system by 30% and 43%, respectively, indicating higher deamination rates and greater arginine consumption. The toluene-exposed cells exhibited increased 15N abundances of protein-bound amino acids in alanine, aspartic acid, glutamic acid, and tyrosine. Furthermore, structural analyses revealed the presence of N[Omega]-acetylarginine and pyrrolidonecarboxylic acid--compounds that had not previously been detected in Tetrahymena pyriformis. Differences in the 15N-enrichment of free amino acids were also evident. This new effect-monitoring system designed to investigate the impact of a pollutant on protein metabolism by using a stable isotope-labeled cell culture is a powerful tool for environmental medical research. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9681977

  3. (15)N investigation of nitrogen released from tobacco-waste to be utilized by maize crop.

    PubMed

    Karaman, M Rüştü; Brohi, A Reşit; Inal, Ali; Aydeniz, Akgün

    2004-12-01

    The (15)N study aimed to estimate the portion of nitrogen released from tobacco-waste to be utilized by maize crop. Tobacco-waste at the levels of 0, 2, 4, 6 and 8 g pot(-1) and ((15)NH(4))(2)SO(4) as nitrogen fertilizer labelled with 5 at.% exc. at the levels of 0, 4, 8, 12 and 16 mg N pot(-1) together with a basal dressing of some nutrients were added to pots with Pioneer maize variety. After the harvest, dry matter yield was recorded and (15)N determinations and calculations were made. Tobacco-waste had a positive effect on the growth and on the nitrogen uptake of maize crop. Increasing the rates of tobacco-waste increased the dry matter yield of maize crop from 4.64 g pot(-1) (at control) to 7.22 g pot(-1) (at the tobacco-waste treatment of 8 g pot(-1)). The values of (15)N in the plant derived from nitrogenous fertilizer also increased with increasing nitrogen fertilizer levels, whereas they decreased from 0.427 % to 0.249 % with increasing tobacco-waste treatments from 0 to 8 g pot(-1), respectively. The average values of per cent nitrogen derived from nitrogenous fertilizer (Ndff) varied from 4.32 % to 7.95 % at the rates of 4-16 mg N pot(-1), respec-tively. However, Ndff decreased from 8.54 % to 4.99 % with increasing tobacco-waste treatments from 0 to 8 g pot(-1), respectively. Per cent nitrogen derived from tobacco-waste (Ndft) increased from 21.8 % to 38.5 % with increasing tobacco-waste treatments from 2 to 8 g pot(-1), respectively. The results have revealed that (15)N tracer technique was confidently used for the investigation of nitrogen levels released from tobacco-waste as organic waste to be utilized by maize crop.

  4. Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance.

    PubMed Central

    Bak, M; Bywater, R P; Hohwy, M; Thomsen, J K; Adelhorst, K; Jakobsen, H J; Sørensen, O W; Nielsen, N C

    2001-01-01

    The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel. PMID:11509381

  5. Steroselective synthesis and application of L-( sup 15 N) amino acids

    SciTech Connect

    Unkefer, C.J. ); Lodwig, S.N. . Div. of Science)

    1991-01-01

    We have developed two general approaches to the stereoselective synthesis of {sup 15}N- and {sup 13}C-labeled amino acids. First, labeled serine, biosynthesized using the methylotrophic bacterium M. extorquens AM1, serves as a chiral precursor for the synthesis of other amino acids. For example, pyridoxal phosphate enzymes can be used for the conversion of L-({alpha}-{sup 15}N)serine to L-({alpha}-{sup 15}N)tyrosine, L-({alpha}-{sup 15}N)tryptophan, and L-({alpha}-{sup 15}N)cysteine. In the second approach, developed by Oppolzer and Tamura, an electrophilic amination'' reagent, 1-chloro-1-nitrosocyclohexane, was used to convert chiral enolates into L-{alpha}-amino acids. We prepared 1-chloro-1-({sup 15}N) nitrosocyclohexane and used it to aminate chiral enolates to produce L-({alpha}-{sup 15}N)amino acids. The stereoselectivity of this scheme using the Oppolzer sultam chiral auxiliary is remarkable, producing enantiomer ratios of 200 to 1. 22 refs., 4 figs.

  6. 15N fractionation in infrared-dark cloud cores

    NASA Astrophysics Data System (ADS)

    Zeng, S.; Jiménez-Serra, I.; Cosentino, G.; Viti, S.; Barnes, A. T.; Henshaw, J. D.; Caselli, P.; Fontani, F.; Hily-Blant, P.

    2017-07-01

    Context. Nitrogen is one of the most abundant elements in the Universe and its 14N/15N isotopic ratio has the potential to provide information about the initial environment in which our Sun formed. Recent findings suggest that the solar system may have formed in a massive cluster since the presence of short-lived radioisotopes in meteorites can only be explained by the influence of a supernova. Aims: We seek to determine the 14N/15N ratio towards a sample of cold and dense cores at the initial stages in their evolution. Methods: We observed the J = 1 → 0 transitions of HCN, H13CN, HC15N, HN13C, and H15NC towards a sample of 22 cores in four infrared-dark clouds (IRDCs) which are believed to be the precursors of high-mass stars and star clusters. Assuming LTE and a temperature of 15 K, the column densities of HCN, H13CN, HC15N, HN13C, and H15NC are calculated and their 14N/15N ratio is determined for each core. Results: The 14N/15N ratios measured in our sample of IRDC cores range between 70 and ≥763 in HCN and between 161 and 541 in HNC. These ratios are consistent with the terrestrial atmosphere (TA) and protosolar nebula (PSN) values, and with the ratios measured in low-mass prestellar cores. However, the 14N/15N ratios measured in cores C1, C3, F1, F2, and G2 do not agree with the results from similar studies towards the same cores using nitrogen bearing molecules with nitrile functional group (-CN) and nitrogen hydrides (-NH) although the ratio spread covers a similar range. Conclusions: Relatively low 14N/15N ratios amongst the four-IRDCs were measured in IRDC G which are comparable to those measured in small cosmomaterials and protoplanetary disks. The low average gas density of this cloud suggests that the gas density, rather than the gas temperature, may be the dominant parameter influencing the initial nitrogen isotopic composition in young PSN. The reduced spectra (FITS files) are only available at the CDS via anonymous ftp to http

  7. NMR study of non-structural proteins--part II: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV).

    PubMed

    Makrynitsa, Garyfallia I; Ntonti, Dioni; Marousis, Konstantinos D; Tsika, Aikaterini C; Lichière, Julie; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-10-01

    Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/β-fold.

  8. Electron-microscopic study of the structure of the surface layer in high-nitrogen 05Kh22AG15N8M2F steel after face turning

    NASA Astrophysics Data System (ADS)

    Blinov, E. V.

    2016-01-01

    The structure of the surface layer in high-nitrogen 05Kh22AG15N8M2F steel workpieces subjected to face turning is studied by electron microscopy. It is found that improved machinability by VK8 alloy cutting tools is achieved at a cutting depth of 0.25 mm and that the cutting-tool life decreases sharply when the cutting depth increases to 1 mm. A nanocrystalline structure with nanocrystal sizes from several to several tens of nanometers forms in the surface layer upon face turning in the as-cast, hot-rolled, and thermally deformed states. The structure of the surface layer is characterized by a high dislocation density and large austenite fragments with broad subgrains and deformation twins.

  9. A novel method for determination of the (15) N isotopic composition of Rubisco in wheat plants exposed to elevated atmospheric carbon dioxide.

    PubMed

    Aranjuelo, Iker; Molero, Gemma; Avice, Jean Christophe; Bourguignon, Jacques

    2015-02-01

    Although ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is mostly known as a key enzyme involved in CO2 assimilation during the Calvin cycle, comparatively little is known about its role as a pool of nitrogen storage in leaves. For this purpose, we developed a protocol to purify Rubisco that enables later analysis of its (15) N isotope composition (δ(15) N) at the natural abundance and (15) N-labeled plants. In order to test the utility of this protocol, durum wheat (Triticum durum var. Sula) exposed to an elevated CO2 concentration (700 vs 400 µmol mol(-1) ) was labeled with K(15) NO3 (enriched at 2 atom %) during the ear development period. The developed protocol proves to be selective, simple, cost effective and reproducible. The study reveals that (15) N labeling was different in total organic matter, total soluble protein and the Rubisco fraction. The obtained data suggest that photosynthetic acclimation in wheat is caused by Rubisco depletion. This depletion may be linked to preferential nitrogen remobilization from Rubisco toward grain filling.

  10. (15)N CSA tensors and (15)N-(1)H dipolar couplings of protein hydrophobic core residues investigated by static solid-state NMR.

    PubMed

    Vugmeyster, Liliya; Ostrovsky, Dmitry; Fu, Riqiang

    2015-10-01

    In this work, we assess the usefulness of static (15)N NMR techniques for the determination of the (15)N chemical shift anisotropy (CSA) tensor parameters and (15)N-(1)H dipolar splittings in powder protein samples. By using five single labeled samples of the villin headpiece subdomain protein in a hydrated lyophilized powder state, we determine the backbone (15)N CSA tensors at two temperatures, 22 and -35 °C, in order to get a snapshot of the variability across the residues and as a function of temperature. All sites probed belonged to the hydrophobic core and most of them were part of α-helical regions. The values of the anisotropy (which include the effect of the dynamics) varied between 130 and 156 ppm at 22 °C, while the values of the asymmetry were in the 0.32-0.082 range. The Leu-75 and Leu-61 backbone sites exhibited high mobility based on the values of their temperature-dependent anisotropy parameters. Under the assumption that most differences stem from dynamics, we obtained the values of the motional order parameters for the (15)N backbone sites. While a simple one-dimensional line shape experiment was used for the determination of the (15)N CSA parameters, a more advanced approach based on the "magic sandwich" SAMMY pulse sequence (Nevzorov and Opella, 2003) was employed for the determination of the (15)N-(1)H dipolar patterns, which yielded estimates of the dipolar couplings. Accordingly, the motional order parameters for the dipolar interaction were obtained. It was found that the order parameters from the CSA and dipolar measurements are highly correlated, validating that the variability between the residues is governed by the differences in dynamics. The values of the parameters obtained in this work can serve as reference values for developing more advanced magic-angle spinning recoupling techniques for multiple labeled samples.

  11. 15N CSA tensors and 15N-1H dipolar couplings of protein hydrophobic core residues investigated by static solid-state NMR

    NASA Astrophysics Data System (ADS)

    Vugmeyster, Liliya; Ostrovsky, Dmitry; Fu, Riqiang

    2015-10-01

    In this work, we assess the usefulness of static 15N NMR techniques for the determination of the 15N chemical shift anisotropy (CSA) tensor parameters and 15N-1H dipolar splittings in powder protein samples. By using five single labeled samples of the villin headpiece subdomain protein in a hydrated lyophilized powder state, we determine the backbone 15N CSA tensors at two temperatures, 22 and -35 °C, in order to get a snapshot of the variability across the residues and as a function of temperature. All sites probed belonged to the hydrophobic core and most of them were part of α-helical regions. The values of the anisotropy (which include the effect of the dynamics) varied between 130 and 156 ppm at 22 °C, while the values of the asymmetry were in the 0.32-0.082 range. The Leu-75 and Leu-61 backbone sites exhibited high mobility based on the values of their temperature-dependent anisotropy parameters. Under the assumption that most differences stem from dynamics, we obtained the values of the motional order parameters for the 15N backbone sites. While a simple one-dimensional line shape experiment was used for the determination of the 15N CSA parameters, a more advanced approach based on the ;magic sandwich; SAMMY pulse sequence (Nevzorov and Opella, 2003) was employed for the determination of the 15N-1H dipolar patterns, which yielded estimates of the dipolar couplings. Accordingly, the motional order parameters for the dipolar interaction were obtained. It was found that the order parameters from the CSA and dipolar measurements are highly correlated, validating that the variability between the residues is governed by the differences in dynamics. The values of the parameters obtained in this work can serve as reference values for developing more advanced magic-angle spinning recoupling techniques for multiple labeled samples.

  12. Measuring denitrification after grassland renewal and grassland conversion to cropland by using the 15N gas-flux method

    NASA Astrophysics Data System (ADS)

    Buchen, Caroline; Eschenbach, Wolfram; Flessa, Heinz; Giesemann, Anette; Lewicka-Szczebak, Dominika; Well, Reinhard

    2015-04-01

    Denitrification, the reduction of oxidized forms of inorganic N to N2O and N2 is an important pathway of gaseous nitrogen losses. Measuring denitrification, especially the reduction of N2O to N2, expressed in the product ratio (N2O/(N2O + N2)), is rather difficult and hence rarely performed under field conditions. But using the 15N gas-flux method allows determining N transformation processes in their natural environment. In order to develop effective climate mitigation strategies understanding the N2O source is essential. We used the 15N gas-flux method to determine N2O and N2 emissions following grassland renewal and conversion techniques. Therefore we selected three different treatments: control (C), mechanical grassland renovation (GR) (autumn 2013) and grassland conversion to maize (GM) (spring 2014) from field plot trials on two different sites (Histic Gleysoil and Plaggic Anthrosol) near Oldenburg, Lower Saxony, Germany. We applied 15N labeled KNO3- (60 atom. % 15N) at a rate equivalent to common farming practices (150 kg N*ha-1) using needle injection of fertilizer solution in three different depths (10 cm, 15 cm, 20 cm) for homogeneous soil labeling up to 30 cm in microplots. During the first 10 days after application (May 2014) gas flux measurements from closed chambers were performed every second day and then weekly following a period of 8 weeks. Gas samples were analyzed for δ15N of N2 and N2O by IRMS according to Lewicka-Szczebak et al. (2013). Concentration and 15N enrichment of NO3- in soil water was determined on weekly samples using the SPIN-MAS technique (Stange et al. 2007). Fluxes of N2 and N2O evolved from the 15N labeled soil nitrogen pool were calculated using the equations of Spott et al. (2006). Peak events of N2 and N2O emissions occurred during the first 10 days of measurement, showing differences in soil types, as well as treatment variations. N2 fluxes up to 178 g*ha-1*day-1 and N2O fluxes up to 280 g*ha-1*day-1 were measured on the

  13. Isotope-Labeled Amyloids via Synthesis, Expression, and Chemical Ligation for Use in FTIR, 2D IR, and NMR Studies.

    PubMed

    Zhang, Tianqi O; Grechko, Maksim; Moran, Sean D; Zanni, Martin T

    2016-01-01

    This chapter provides protocols for isotope-labeling the human islet amyloid polypeptide (hIAPP or amylin) involved in type II diabetes and γD-crystallin involved in cataract formation. Because isotope labeling improves the structural resolution, these protocols are useful for experiments using Fourier transform infrared (FTIR), two-dimensional infrared (2D IR), and NMR spectroscopies. Our research group specializes in using 2D IR spectroscopy and isotope labeling. 2D IR spectroscopy provides structural information by measuring solvation from 2D diagonal lineshapes and vibrational couplings from cross peaks. Infrared spectroscopy can be used to study kinetics, membrane proteins, and aggregated proteins. Isotope labeling provides greater certainty in the spectral assignment, which enables new structural insights that are difficult to obtain with other methods. For amylin, we provide a protocol for (13)C/(18)O labeling backbone carbonyls at one or more desired amino acids in order to obtain residue-specific structural resolution. We also provide a protocol for expressing and purifying amylin from E. coli, which enables uniform (13)C or (13)C/(15)N labeling. Uniform labeling is useful for measuring the monomer infrared spectrum in an amyloid oligomer or fiber as well as amyloid protein bound to another polypeptide or protein, such as a chaperone or an inhibitor. In addition, our expression protocol results in 2-2.5 mg of amylin peptide per 1 L cell culture, which is a high enough yield to straightforwardly obtain the 2-10 mg needed for high resolution and solid-state NMR experiments. Finally, we provide a protocol to isotope-label either of the two domains of γD-crystallin using expressed protein ligation. Domain labeling makes it possible to resolve the structures of the two halves of the protein in FTIR and 2D IR spectra. With modifications, these strategies and protocols for isotope labeling can be applied to other amyloid polypeptides and proteins.

  14. Novel tracer method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies.

    PubMed

    Wu, Dianming; Kampf, Christopher J; Pöschl, Ulrich; Oswald, Robert; Cui, Junfang; Ermel, Michael; Hu, Chunsheng; Trebs, Ivonne; Sörgel, Matthias

    2014-07-15

    Gaseous nitrous acid (HONO), the protonated form of nitrite, contributes up to ∼60% to the primary formation of hydroxyl radical (OH), which is a key oxidant in the degradation of most air pollutants. Field measurements and modeling studies indicate a large unknown source of HONO during daytime. Here, we developed a new tracer method based on gas-phase stripping-derivatization coupled to liquid chromatography-mass spectrometry (LC-MS) to measure the 15N relative exceedance, ψ(15N), of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye, purified by solid phase extraction (SPE), and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In the optimal working range of ψ(15N)=0.2-0.5, the relative standard deviation of ψ(15N) is <4%. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method was applied to measure HO15NO emissions from soil in a dynamic chamber with and without spiking 15) labeled urea. The identification of HO15NO from soil with 15N urea addition confirmed biogenic emissions of HONO from soil. The method enables a new approach of studying the formation pathways of HONO and its role for atmospheric chemistry (e.g., ozone formation) and environmental tracer studies on the formation and conversion of gaseous HONO or aqueous NO2- as part of the biogeochemical nitrogen cycle, e.g., in the investigation of fertilization effects on soil HONO emissions and microbiological conversion of NO2- in the hydrosphere.

  15. Synthesis of isotopically labeled daclatasvir for use in human clinical studies.

    PubMed

    Easter, John A; Burrell, Richard C; Bonacorsi, Samuel J

    2016-04-01

    Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol-Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon-14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 μCi/mg. To support a human absolute bioavailability study, 5.56 g of [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Stem injection of 15N-NH4NO3 into mature Sitka spruce (Picea sitchensis).

    PubMed

    Nair, Richard; Weatherall, Andrew; Perks, Mike; Mencuccini, Maurizio

    2014-10-01

    Stem injection techniques can be used to introduce (15)N into trees to overcome a low variation in natural abundance and label biomass with a distinct (15)N signature, but have tended to target small and young trees, of a variety of species, with little replication. We injected 98 atom% (15)N ammonium nitrate (NH4NO3) solution into 13 mature, 9- to 13-m tall edge-profile Sitka spruce trees in order to produce a large quantity of labelled litter, examining the distribution of the isotope throughout the canopy after felling in terms of both total abundance of (15)N and relative distribution of the isotope throughout individual trees. Using a simple mass balance of the canopy alone, based on observed total needle biomass and modelled branch biomass, all of the isotope injected was accounted for, evenly split between needles and branches, but with a high degree of variability both within individual trees, and among trees. Both (15)N abundance and relative within-canopy distribution were biased towards the upper and middle crown in foliage. Recovery of the label in branches was much more variable than in needles, possibly due to differences in nitrogen allocation for both growth and storage, which differ seasonally between foliage and woody biomass.

  17. Uptake and fate of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in coastal marine biota determined using a stable isotopic tracer, (15)N - [RDX].

    PubMed

    Ballentine, Mark L; Ariyarathna, Thivanka; Smith, Richard W; Cooper, Christopher; Vlahos, Penny; Fallis, Stephen; Groshens, Thomas J; Tobias, Craig

    2016-06-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is globally one of the most commonly used military explosives and environmental contaminant. (15)N labeled RDX was added into a mesocosm containing 9 different coastal marine species in a time series experiment to quantify the uptake of RDX and assess the RDX derived (15)N retention into biota tissue. The (15)N attributed to munitions compounds reached steady state concentrations ranging from 0.04 to 0.67 μg (15)N g dw(-1), the bulk (15)N tissue concentration for all species was 1-2 orders of magnitude higher suggesting a common mechanism or pathway of RDX biotransformation and retention of (15)N. A toxicokinetic model was created that described the (15)N uptake, elimination, and transformation rates. While modeled uptake rates were within previous published values, elimination rates were several orders of magnitude smaller than previous studies ranging from 0.05 to 0.7 days(-1). These small elimination rates were offset by high rates of retention of (15)N previously not measured. Bioconcentration factors and related aqueous:organism ratios of compounds and tracer calculated using different tracer and non-tracer methods yielded a broad range of values (0.35-101.6 mL g(-1)) that were largely method dependent. Despite the method-derived variability, all values were generally low and consistent with little bioaccumulation potential. The use of (15)N labeled RDX in this study indicates four possible explanations for the observed distribution of compounds and tracer; each with unique potential implications for possible toxicological impacts in the coastal marine environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Dynamic spin label study of the barstar-barnase complex.

    PubMed

    Timofeev, V P; Balandin, T G; Tkachev, Ya V; Novikov, V V; Lapuk, V A; Deev, S M

    2007-09-01

    The dynamic spin label method was used to study protein-protein interactions in the model complex of the enzyme barnase (Bn) with its inhibitor barstar. The C40A mutant of barstar (Bs) containing a single cysteine residue was modified with two different spin labels varying in length and structure of a flexible linker. Each spin label was selectively bound to the Cys82 residue, located near the Bn-Bs contact site. The formation of the stable protein complex between Bn and spin labeled Bs was accompanied by a substantial restriction of spin label mobility, indicated by remarkable changes in the registered EPR spectra. Order parameter, S, as an estimate of rapid reorientation of spin label relative to protein molecule, was sharply increasing approaching 1. However, the rotational correlation time tau for spin-labeled Bs and its complex with Bn in solution corresponded precisely to their molecular weights. These data indicate that both Bs and its complex with Bn are rigid protein entities. Spin labels attached to Bs in close proximity to an interface of interaction with Bn, regardless of its structure, undergo significant restriction of mobility by the environment of the contact site of the two proteins. The results show that this approach can be used to investigate fusion proteins containing Bn or Bs.

  19. Production of isotopically-labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies

    PubMed Central

    Gómez-Cortés, Pilar; Brenna, J. Thomas; Sacks, Gavin L.

    2012-01-01

    Optimal accuracy and precision in small molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time consuming, and many labeled compounds are not available in pure form. We used a single prototype uniformly labeled [U-13C]-compound to generate [U-13C]-volatile standards for use in subsequent experimental profiling studies. [U-13C]-α-linolenic acid (C18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-13C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography-time of flight-mass spectrometry (HS-SPME-GC-TOF-MS) by comparison of spectra with unlabeled volatiles. Using 250 μL starting sample, labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-13C]-labeled standards, limits of detection comparable to or better than previous HS-SPME reports were achieved, 0.010–1.04 ng/g. The performance of the [U-13C]-volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60°C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-13C]-oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-13C]-standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-13C]-sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to

  20. Release of nitrous oxide and dinitrogen from a transition bog under drained and rewetted conditions due to denitrification: results from a [15N]nitrate-bromide double-tracer study.

    PubMed

    Tauchnitz, Nadine; Spott, Oliver; Russow, Rolf; Bernsdorf, Sabine; Glaser, Bruno; Meissner, Ralph

    2015-01-01

    Denitrification is well known being the most important nitrate-consuming process in water-logged peat soils, whereby the intermediate compound nitrous oxide (N(2)O) and the end product dinitrogen (N(2)) are ultimately released. The present study was aimed at evaluating the release of these gases (due to denitrification) from a nutrient-poor transition bog ecosystem under drained and three differently rewetted conditions at the field scale using a (15)N-tracer approach ([(15)N]nitrate application, 30 kg N ha(-1)) and a common closed-chamber technique. The drained site is characterized by a constant water table (WT) of -30 cm (here referred to as D30), while rewetted sites represent a constant WT of -15 cm, a constant WT of 0 cm (i.e. waterlogged), and an initial WT of 0 cm (which decreased slightly during the experiment), respectively, (here referred to as R15, R0, and R0(d), respectively). The highest N(2)O emissions were observed at D30 (291 µg N(2)O-N m(-2) h(-1)) as well as at R0d (665 µg N(2)O-N m(-2) h(-1)). At the rewetted peat sites with a constant WT (i.e. R15 and R0), considerably lower N2O emissions were observed (maximal 37 µg N(2)O-N m(-2) h(-1)). Concerning N(2) only at the initially water-logged peat site R0d considerable release rates (up to 3110 µg N(2)-N m(-2) h(-1)) were observed, while under drained conditions (D30) no N(2) emission and under rewetted conditions with a constant WT (R15 and R0) significantly lower N(2) release rates (maximal 668 µg N(2)-N m(-2) h(-1)) could be detected. In addition, it has been found that natural WT fluctuations at rewetted peat sites, in particular a rapid drop down of the WT, can induce high emission rates for both N(2)O and N(2).

  1. The effectiveness of auxiliary prescription labels: a pilot study.

    PubMed

    Wiederholt, J B; Kotzan, J A; Cooper, J W

    1983-03-01

    Affixed auxiliary prescription labels are widely used in the practice of pharmacy because they supposedly provide the patient with pertinent information that is not contained within the prescription signature. Yet, whether the labels are effective is not known, nor is it known whether the label's elements, such as color, form, and logo, affect perception of the written text. Sound scientific analyses of these questions are limited. Therefore, a pilot study involving a series of experiments was designed to determine whether individual perception of pertinent information is affected by the use of affixed auxiliary prescription labels. The second objective of this study was to evaluate how color and logo differences affected perception of the label's written text. Participants were selected for the experiments after being screened for color blindness, corrected vision, and, in some cases, previous pharmacy employment. Subjects viewed labels affixed to prescription vials via a two-channel tachistoscope. The tachistoscopic methodology measured perception, and its accuracy was verified through a forced-choice instrument. Results from the pilot study were threefold: (1) a sound scientific analysis found affixed auxiliary labels to be effective, (2) significant variance could be attributed to both individual and subject differences, and (3) the unique effects of color and logo could not be determined.

  2. Dynamics of 4-oxo-TEMPO-d16-15N nitroxide-propylene glycol system studied by ESR and ESE in liquid and glassy state in temperature range 10-295 K

    NASA Astrophysics Data System (ADS)

    Goslar, Janina; Hoffmann, Stanislaw K.; Lijewski, Stefan

    2016-08-01

    ESR spectra and electron spin relaxation of nitroxide radical in 4-oxo-TEMPO-d16-15N in propylene glycol were studied at X-band in the temperature range 10-295 K. The spin-lattice relaxation in the liquid viscous state determined from the resonance line shape is governed by three mechanisms occurring during isotropic molecular reorientations. In the glassy state below 200 K the spin-lattice relaxation, phase relaxation and electron spin echo envelope modulations (ESEEM) were studied by pulse spin echo technique using 2-pulse and 3-pulse induced signals. Electron spin-lattice relaxation is governed by a single non-phonon relaxation process produced by localized oscillators of energy 76 cm-1. Electron spin dephasing is dominated by a molecular motion producing a resonance-type peak in the temperature dependence of the dephasing rate around 120 K. The origin of the peak is discussed and a simple method for the peak shape analysis is proposed, which gives the activation energy of a thermally activated motion Ea = 7.8 kJ/mol and correlation time τ0 = 10-8 s. The spin echo amplitude is strongly modulated and FT spectrum contains a doublet of lines centered around the 2D nuclei Zeeman frequency. The splitting into the doublet is discussed as due to a weak hyperfine coupling of nitroxide unpaired electron with deuterium of reorienting CD3 groups.

  3. Nitrate Reduction in a Groundwater Microcosm Determined by 15N Gas Chromatography-Mass Spectrometry

    PubMed Central

    Bengtsson, Göran; Annadotter, Heléne

    1989-01-01

    Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO3−-N liter−1. Traces of 15NO3− were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day−1. Both assimilatory and dissimilatory reduction rates were estimated from analyses of 15N2, 15N2O, 15NH4+, and 15N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N2 and N2O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO3− and NH4+ were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their [methyl-3H]thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g−1 day−1. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N2, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO3− was reduced to NH4+. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month. PMID:16348048

  4. The impact of the pi-electron conjugation on (15)N, (13)C and (1)H NMR chemical shifts in push-pull benzothiazolium salts. Experimental and theoretical study.

    PubMed

    Hrobárik, Peter; Horváth, Branislav; Sigmundová, Ivica; Zahradník, Pavol; Malkina, Olga L

    2007-11-01

    The (15)N as well as (13)C and (1)H chemical shifts of eight push-pull benzothiazolium iodides with various pi-conjugated chains between dimethylamino group and benzothiazolium moiety have been determined by NMR spectroscopy at the natural-abundance level of all nuclei in DMSO-d(6) solution. In general, the quaternary benzothiazolium nitrogen is more shielded [delta((15)N-3) vary between - 241.3 and - 201.9 ppm] with respect to parent 3-methylbenzothiazolium iodide [delta((15)N-3) = - 183.8 ppm], depending on the length and constitution of the pi-conjugated bridge. A larger variation in (15)N chemical shifts is observed on dimethylamino nitrogen, which covers the range of - 323.3 to - 257.2 ppm. The effect of pi-conjugation degree has a less pronounced influence on (13)C and (1)H chemical shifts. Experimental data are interpreted by means of density functional theory (DFT) calculations. Reasonable agreement between theoretical and experimental (15)N NMR chemical shifts was found, particularly when performing calculations with hybrid exchange-correlation functionals. A better accord with experiment is achieved by utilizing a polarizable continuum model (PCM) along with an explicit treatment of hydrogen-bonding between the solute and the water present in dimethylsulfoxide (DMSO). Finally, (13)C and (1)H NMR spectra were computed and analysed in order to compare them with available experimental data. (c) 2007 John Wiley & Sons, Ltd.

  5. Soil processes drive seasonal variation in retention of 15N tracers in a deciduous forest catchment.

    PubMed

    Goodale, Christine L; Fredriksen, Guinevere; Weiss, Marissa S; McCalley, K; Sparks, Jed P; Thomas, Steven A

    2015-10-01

    Seasonal patterns of stream nitrate concentration have long been interpreted as demonstrating the central role of plant uptake in regulating stream nitrogen loss from forested catchments. Soil processes are rarely considered as important drivers of these patterns. We examined seasonal variation in N retention in a deciduous forest using three whole-ecosystem 15N tracer additions: in late April (post-snowmelt, pre-leaf-out), late July (mid-growing- season), and late October (end of leaf-fall). We expected that plant 15N uptake would peak in late spring and midsummer, that immobilization in surface litter and soil would peak the following autumn leaf-fall, and that leaching losses would vary inversely with 15N retention. Similar to most other 15N tracer studies, we found that litter and soils dominated ecosystem retention of added 15N. However, 15N recovery in detrital pools varied tremendously by season, with > 90% retention in spring and autumn and sharply reduced 15N retention in late summer. During spring, over half of the 15N retained in soil occurred within one day in the heavy (mineral-associated) soil fraction. During summer, a large decrease in 15N retention one week after addition coincided with increased losses of 15NO3- to soil leachate and seasonal increases in soil and stream NO3- concentrations, although leaching accounted for only a small fraction of the lost 15N (< 0.2%). Uptake of 15N into roots did not vary by season and accounted for < 4% of each tracer addition. Denitrification or other processes that lead to N gas loss may have consumed the rest. These measurements of 15N movement provide strong evidence for the dominant role of soil processes in regulating seasonal N retention and losses in this catchment and perhaps others with similar soils.

  6. Reconstitution and electron spin resonance spin labeling studies of nucleosomes

    SciTech Connect

    Chan, D.C.F.; Grover, T.A.; Piette, L.H.

    1980-10-01

    The spin label, N-(2,2,5,5-tetramethyl-3-carbonylpyrrolidine-1-oxyl)-imidazole, was used to study the mode of reconstitution of nucleosome core particles. The histone cores in 2 M NaCl were first reacted with the imidazole spin label. After the removal of unreacted label, the histone cores were mixed with purified core DNA (145 base pairs) in 2 M NaCl. The mixture was then reconstituted by salt step-gradient dialysis according to Tatchell and Van Holde. At each step of the dialysis, an electron spin resonance (ESR) spectrum of the labeled tyrosyls was recorded and the correlation time of the label determined. As the ionic strength was gradually decreased, the correlation time of the spin label increased. This is in contrast to what we observed previously for the histone core alone, in which a decrease in the ionic strength caused the histone core (in the absence of DNA) to dissociate, freeing up the label and decreasing its correlation tie. Judging from the change in rotational correlation times for the spin label, we concluded that the histone core binds progressively to the DNA in the range of 2 M-0.3 M NaCl. When the ionic strength is <0.3 M, full association between the histone core and DNA takes place. These reconstituted spin labelled nucleosome core complexes, purified by isokinetic sucrose gradient, were found to have identical physical properties (histone content, sedimentation coefficient, thermal melting profile, and ciruclar dichroism) as the native particle.

  7. Influence of N-H...O and O-H...O hydrogen bonds on the (17)O, (15)N and (13)C chemical shielding tensors in crystalline acetaminophen: a density functional theory study.

    PubMed

    Esrafili, Mehdi D; Behzadi, Hadi; Hadipour, Nasser L

    2007-06-01

    A computational investigation was carried out to characterize the (17)O, (15)N and (13)C chemical shielding tensors in crystalline acetaminophen. We found that N-H...O and O-H...O hydrogen bonds around the acetaminophen molecule in the crystal lattice have different influences on the calculated (17)O, (15)N and (13)C chemical shielding eigenvalues and their orientations in the molecular frame of axes. The calculations were performed with the B3LYP method and 6-311++G(d, p) and 6-311+G(d) standard basis sets using the Gaussian 98 suite of programs. Calculated chemical shielding tensors were used to evaluate the (17)O, (15)N, and (13)C NMR chemical shift tensors in crystalline acetaminophen, which are in reasonable agreement with available experimental data. The difference between the calculated NMR parameters of the monomer and molecular clusters shows how much hydrogen-bonding interactions affect the chemical shielding tensors of each nucleus. The computed (17)O chemical shielding tensor on O(1), which is involved in two intermolecular hydrogen bonds, shows remarkable sensitivity toward the choice of the cluster model, whereas the (17)O chemical shielding tensor on O(2) involved in one N-H...O hydrogen bond, shows smaller improvement toward the hydrogen-bonding interactions. Also, a reasonably good agreement between the experimentally obtained solid-state (15)N and (13)C NMR chemical shifts and B3LYP/6-311++G(d, p) calculations is achievable only in molecular cluster model where a complete hydrogen-bonding network is considered. Moreover, at the B3LYP/6-311++G(d, p) level of theory, the calculated (17)O, (15)N and (13)C chemical shielding tensor orientations are able to reproduce the experimental values to a reasonably good degree of accuracy.

  8. Isotope effect study of {kappa}-(BEDT-TTF){sub 2}Cu(NCS){sub 2}: Labeling in the anion

    SciTech Connect

    Kini, A.M.; Wang, H.H.; Schlueter, J.A.

    1995-12-31

    Since the initial discovery of organic superconductivity in 1979, a large number of organic superconductors have now been synthesized. However, the mechanism of electron-pairing in these novel superconductors has remained largely unresolved. Isotope effect studies constitute an important experimental tool for the investigation of whether or not the electron-pairing mechanism in organic superconductors is phonon-mediated, as in conventional superconductors. Recent isotope effect studies in the authors` laboratory, involving seven different isotopically labeled BEDT-TTF (or ET) derivatives, have demonstrated the following: (1) intramolecular phonon modes involving C{double_bond}C and C{single_bond}S stretching vibrations in the ET donor molecule are not the dominant mediators of electron-pairing, and (2) in {kappa}-(ET){sub 2}Cu(NCS){sub 2}, there exist two competing isotope effects--a normal mass effect, i.e., lowering of {Tc} upon isotopic labeling, when the ET molecular mass is increased by concurrent {sup 13}C and {sup 34}S labeling, in addition to an inverse isotope effect upon deuterium labeling in ET. It is of great interest to investigate if there is an isotope effect when the charge-compensating anions, which are also located within the non-conducting layer in the superconducting cation-radical salts, are isotopically labeled. The existence of an isotope effect when the anions are labeled would be indicative of electron-pairing with the mediation of vibrational frequencies associated with the anions. In this paper, the authors present the results of the first isotope effect study in which isotopic labeling in the anion portion of {kappa}-(ET){sub 2}Cu(NCS){sub 2} is carried out. The authors find no isotope effect when the carbon and nitrogen atoms of the thiocyanate groups in the anion are replaced with {sup 13}C and {sup 15}N isotopes.

  9. Study of conformations and hydrogen bonds in the configurational isomers of pyrrole-2-carbaldehyde oxime by 1H, 13C and 15N NMR spectroscopy combined with MP2 and DFT calculations and NBO analysis.

    PubMed

    Afonin, Andrei V; Ushakov, Igor A; Pavlov, Dmitry V; Ivanov, Andrei V; Mikhaleva, Al'bina I

    2010-09-01

    The (1)H, (13)C and (15)N NMR studies have shown that the E and Z isomers of pyrrole-2-carbaldehyde oxime adopt preferable conformation with the syn orientation of the oxime group with respect to the pyrrole ring. The syn conformation of E and Z isomers of pyrrole-2-carbaldehyde oxime is stabilized by the N-H...N and N-H...O intramolecular hydrogen bonds, respectively. The N-H...N hydrogen bond in the E isomer causes the high-frequency shift of the bridge proton signal by about 1 ppm and increase the (1)J(N, H) coupling by approximately 3 Hz. The bridge proton shows further deshielding and higher increase of the (1)J(N, H) coupling constant due to the strengthening of the N-H...O hydrogen bond in the Z isomer. The MP2 calculations indicate that the syn conformation of E and Z isomers is by approximately 3.5 kcal/mol energetically less favorable than the anti conformation. The calculations of (1)H shielding and (1)J(N, H) coupling in the syn and anti conformations allow the contribution to these constants from the N-H...N and N-H...O hydrogen bondings to be estimated. The NBO analysis suggests that the N-H...N hydrogen bond in the E isomer is a pure electrostatic interaction while the charge transfer from the oxygen lone pair to the antibonding orbital of the N-H bond through the N-H...O hydrogen bond occurs in the Z isomer. 2010 John Wiley & Sons, Ltd.

  10. The structural plasticity of heparan sulfate NA-domains and hence their role in mediating multivalent interactions is confirmed by high-accuracy 15N-NMR relaxation studies

    PubMed Central

    Mobli, Mehdi; Nilsson, Mathias

    2007-01-01

    Considering the biological importance of heparan sulfate (HS) and the significant activity of its highly-sulfated regions (S-domains), the paucity of known functions for the non-sulfated NA-domains is somewhat puzzling. It has been suggested that chain dynamics within the NA-domains are the key to their functional role in HS. In this study, we investigate this hypothesis using state-of-the-art nuclear magnetic resonance (NMR) experiments at multiple frequencies. To resolve the problem of severe overlap in 1H-NMR spectra of repetitive polysaccharides from proteoglycans, we have prepared oligosaccharides with the chemical structure of HS NA-domains containing the 15N nucleus, which has enough chemical shift dispersion to probe the central residues of octasaccharides at atomic resolution using 600 MHz NMR. By performing NMR relaxation experiments at three magnetic-field strengths, high quality data on internal dynamics and rotational diffusion was obtained. Furthermore, translational diffusion could also be measured by NMR using pulse field gradients. These experimental data were used, in concert with molecular dynamics simulations, to provide information on local molecular shape, greatly aiding our relaxation analyses. Our results, which are more accurate than those presented previously, confirm the higher flexibility of the NA-domains as compared with reported data on S-domains. It is proposed that this flexibility has two functional roles. First, it confers a greater area of interaction from the anchoring point on the core protein for the bioactive S-domains. Secondly, it allows multiple interactions along the same HS chain that are dynamically independent of each other. Electronic Supplementary Material The online version of this article (doi:10.1007/s10719-007-9081-9) contains supplementary material, which is available to authorized users. PMID:18080183

  11. Increased Plant Uptake of Nitrogen from 15N Depleted Fertilizer Using Plant Growth-Promoting Rhizobacteria

    USDA-ARS?s Scientific Manuscript database

    The techniques of 15N isotope have been very useful for determining the behavior and fate of N in soil, including the use efficiency of applied N fertilizers by plants. Our objective in this study was to use 15N isotope techniques to demonstrate that a model plant growth-promoting rhizobacteria (PGP...

  12. Compound-specific δ15N amino acid measurements in littoral mussels in the California upwelling ecosystem: a new approach to generating baseline δ15N Isoscapes for coastal ecosystems.

    PubMed

    Vokhshoori, Natasha L; McCarthy, Matthew D

    2014-01-01

    We explored δ(15)N compound-specific amino acid isotope data (CSI-AA) in filter-feeding intertidal mussels (Mytilus californianus) as a new approach to construct integrated isoscapes of coastal primary production. We examined spatial δ(15)N gradients in the California Upwelling Ecosystem (CUE), determining bulk δ(15)N values of mussel tissue from 28 sites between Port Orford, Oregon and La Jolla, California, and applying CSI-AA at selected sites to decouple trophic effects from isotopic values at the base of the food web. Bulk δ(15)N values showed a strong linear trend with latitude, increasing from North to South (from ∼ 7‰ to ∼ 12‰, R(2) = 0.759). In contrast, CSI-AA trophic position estimates showed no correlation with latitude. The δ(15)N trend is therefore most consistent with a baseline δ(15)N gradient, likely due to the mixing of two source waters: low δ(15)N nitrate from the southward flowing surface California Current, and the northward transport of the California Undercurrent (CUC), with (15)N-enriched nitrate. This interpretation is strongly supported by a similar linear gradient in δ(15)N values of phenylalanine (δ(15)NPhe), the best AA proxy for baseline δ(15)N values. We hypothesize δ(15)N(Phe) values in intertidal mussels can approximate annual integrated δ(15)N values of coastal phytoplankton primary production. We therefore used δ(15)N(Phe) values to generate the first compound-specific nitrogen isoscape for the coastal Northeast Pacific, which indicates a remarkably linear gradient in coastal primary production δ(15)N values. We propose that δ(15)N(Phe) isoscapes derived from filter feeders can directly characterize baseline δ(15)N values across major biochemical provinces, with potential applications for understanding migratory and feeding patterns of top predators, monitoring effects of climate change, and study of paleo- archives.

  13. Creating 13C- and 15N-enriched tree leaf litter for decomposition experiments

    NASA Astrophysics Data System (ADS)

    Szlavecz, K. A.; Pitz, S.; Chang, C.; Bernard, M.

    2013-12-01

    at the end of the experiment. Enrichment of roots was significantly higher than leaves (δ13C range: 111.5-219.2‰; δ15N range: 1516.9-3939.3‰) indicating that nutrients were translocated away from leaves prior to senescence, which is supported by the increase in C:N ratio between the initial (19.0) and final (60.1) leaf sampling. Despite the variable levels of enrichment, leaves from all species were sufficiently labeled for use in future studies aimed at tracking the transformation of carbon and nitrogen during decomposition. The greatest challenges were treating diseases and pests and creating ideal growing conditions for many species within the same chamber. Reducing the number of individuals and better pest management will lead to even higher level enrichment in the future.

  14. Allergens labeling on French processed foods - an Oqali study.

    PubMed

    Battisti, Charlène; Chambefort, Amélie; Digaud, Olivier; Duplessis, Barbara; Perrin, Cécile; Volatier, Jean-Luc; Gauvreau-Béziat, Julie; Menard, Céline

    2017-07-01

    The French Observatory of Food Quality (Oqali) aims at collecting all nutritional data provided on labels of processed foods (nutritional information and composition), at branded products level, in order to follow nutritional labeling changes over time. This study carries out an overview of allergens labeling frequencies by distinguishing allergens used in recipes from those listed on precautionary statements, for the fourteen allergen categories for which labeling is mandatory according to European legislation. 17,309 products were collected, between 2008 and 2012, from 26 food categories. Products were classified per family and type of brand (national brands, retailer brands, entry-level retailer brands, hard discount, and specialized retailer brands). Allergenic ingredients were identified from ingredients lists and precautionary statements. 73% of the 17,309 products studied contained at least one allergen in their ingredients list and 39% had a precautionary statement for one or more allergens. Milk (53%), gluten (41%), and egg (22%) were the most commonly used allergens in ingredients lists. For precautionary statement, nuts (20%), egg (14%), peanut (13%), soybean (12%), and milk (11%) were the most common allergens listed. Precautionary statement was most frequently found among first-price products (hard discount and entry-level retailer brands). National brands seemed to use it less frequently. For all these results, differences depended both on food categories and allergen categories. This study will enable to follow allergens labeling and their use as ingredients over time, particularly by assessing an hypothetical increase in allergens presence in processed food.

  15. 3D 15N/15N/1H chemical shift correlation experiment utilizing an RFDR-based 1H/1H mixing period at 100 kHz MAS

    NASA Astrophysics Data System (ADS)

    Nishiyama, Yusuke; Malon, Michal; Ishii, Yuji; Ramamoorthy, Ayyalusamy

    2014-07-01

    Homonuclear correlation NMR experiments are commonly used in the high-resolution structural studies of proteins. While 13C/13C chemical shift correlation experiments utilizing dipolar recoupling techniques are fully utilized under MAS, correlation of the chemical shifts of 15N nuclei in proteins has been a challenge. Previous studies have shown that the negligible 15N-15N dipolar coupling in peptides or proteins necessitates the use of a very long mixing time (typically several seconds) for effective spin diffusion to occur and considerably slows down a 15N/15N correlation experiment. In this study, we show that the use of mixing proton magnetization, instead of 15N, via the recoupled 1H-1H dipolar couplings enable faster 15N/15N correlation. In addition, the use of proton-detection under ultrafast MAS overcomes the sensitivity loss due to multiple magnetization transfer (between 1H and 15N nuclei) steps. In fact, less than 300 nL (∼1.1 micromole quantity) sample is sufficient to acquire the 3D spectrum within 5 h. Our results also demonstrate that a 3D 15N/15N/1H experiment can render higher resolution spectra that will be useful in the structural studies of proteins at ultrafast MAS frequencies. 3D 15N/15N/1H and 2D radio frequency-driven dipolar recoupling (RFDR)-based 1H/1H experimental results obtained from a powder sample of N-acetyla-L-15N-valyl-L-15N-leucine at 70 and 100 kHz MAS frequencies are presented.

  16. Tracking wind-dispersed seeds using (15)N-isotope enrichment.

    PubMed

    Forster, C; Herrmann, J D

    2014-11-01

    Seed dispersal influences a wide range of ecological processes. However, measuring dispersal patterns, particularly long-distance dispersal, has been a difficult task. Marking bird-dispersed seeds with stable (15)N isotopes has been shown to be a user-friendly method to trace seed dispersal. In this study, we determined whether (15)N urea solution could be used to enrich seeds of two common wind-dispersed plants, Eupatorium glaucescens (Asteraceae) and Sericocarpus tortifolius (Asteraceae). We further tested if the water type (distilled versus tap) in (15)N urea solutions influences the level and variability of enrichment of plant seeds, and if increasing spraying frequency per se increases enrichment. Because droughts may lower seed set or kill plants, we wanted to investigate if the additional use of an externally applied anti-transpirant affects the intake of externally applied (15)N into seeds. The results demonstrate that (15)N enrichment of seeds can facilitate dispersal experiments with wind-dispersed plants. The use of distilled water in (15)N urea solutions did not increase (15)N enrichment compared to tap water. Further, enrichment was more efficient at lower spray frequencies. Both the use of tap water and low frequencies could lower time, effort and project costs. The results suggest that species can be protected from drought using an anti-transpirant without decreasing the incorporation of (15)N into seeds. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  17. Nitrate reductase 15N discrimination in Arabidopsis thaliana, Zea mays, Aspergillus niger, Pichea angusta, and Escherichia coli

    PubMed Central

    Carlisle, Eli; Yarnes, Chris; Toney, Michael D.; Bloom, Arnold J.

    2014-01-01

    Stable 15N isotopes have been used to examine movement of nitrogen (N) through various pools of the global N cycle. A central reaction in the cycle involves the reduction of nitrate (NO−3) to nitrite (NO−2) catalyzed by nitrate reductase (NR). Discrimination against 15N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro 15N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic 15N discrimination by the enzyme and to evaluate the validity of measurements made using 15N-enriched NO−3. Observed NR isotope discrimination ranged from 22 to 32‰ (kinetic isotope effects of 1.022–1.032) among the different isozymes at natural abundance 15N (0.37%). As the fractional 15N content of substrate NO−3 increased from natural abundance, the product 15N fraction deviated significantly from that expected based on substrate enrichment and 15N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO−3 and NO−2 in some protocols became a greater source of error as 15N enrichment increased. We briefly discuss potential causes of the experimental artifacts with enriched 15N and recommend against the use of highly enriched 15N tracers to study N discrimination in plants or soils. PMID:25071800

  18. Detection of 2,4,6-Trinitrotoluene-Utilizing Anaerobic Bacteria by 15N and 13C Incorporation ▿

    PubMed Central

    Gallagher, Erin M.; Young, Lily Y.; McGuinness, Lora M.; Kerkhof, Lee J.

    2010-01-01

    2,4,6-Trinitrotoluene (15N or 13C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [15N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [13C]TNT cultures and was related to Lysobacter taiwanensis. PMID:20081008

  19. [Study of erythrocyte dehydration using spin labels].

    PubMed

    Moiseev, V A; Mezhidov, S Kh; Nardid, O A

    1989-01-01

    Possibility of studying erythrocyte dehydration by ESR-spin probe is substantiated. Dehydration of erythrocytes in relation to osmolarity of sodium chloride solutions is investigated. The results are shown to agree with the data obtained by radioisotope method.

  20. X-ray microscopic studies of labeled nuclear cell structures

    NASA Astrophysics Data System (ADS)

    Vogt, S.; Schneider, G.; Steuernagel, A.; Lucchesi, J.; Schulze, E.; Rudolph, D.; Schmahl, G.

    2000-05-01

    In X-ray microscopy different proteins are not readily distinguishable. However, in cell biology it is often desirable to localize single proteins, e.g., inside the cell nucleus. This can be achieved by immunogold labeling. Colloidal gold conjugated antibodies are used to mark the protein specifically. With silver solution these are enlarged so as to heighten their contrast. The strong absorption of silver allows easy visualization of the label in the nuclei. In this study male specific lethal 1 protein in male Drosophila melanogaster cells was labeled. This protein forms, together with four other proteins, a complex that is associated with the male X chromosome. It regulates dosage compensation by enhancing X-linked gene transcription in males. Room temperature and cyro transmission X-ray microscopic images (taken with the Göttingen TXM at BESSY) of these labeled cells are shown. Confocal laser scan microscopy ascertains the correct identification of the label in the X-ray micrographs, and allows comparison of the structural information available from both instruments.

  1. NMR study of non-structural proteins--part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV).

    PubMed

    Melekis, Efstathios; Tsika, Aikaterini C; Lichière, Julie; Chasapis, Christos T; Margiolaki, Irene; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-04-01

    Macro domains are ADP-ribose-binding modules present in all eukaryotic organisms, bacteria and archaea. They are also found in non-structural proteins of several positive strand RNA viruses such as alphaviruses. Here, we report the high yield expression and preliminary structural analysis through solution NMR spectroscopy of the macro domain from New World Mayaro Alphavirus. The recombinant protein was well-folded and in a monomeric state. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure determined by TALOS+.

  2. Conformational changes of plasma fibronectin detected upon adsorption to solid substrates: A spin-label study

    SciTech Connect

    Narasimhan, C.; Lai, Chingsan )

    1989-06-13

    Changes in local environment of the free sulfhydryl groups in plasma fibronectin upon adsorption of the protein to polystyrene beads have been examined by electron spin resonance (ESR) spin-label spectroscopy. The two free sulfhydryl groups per subunit of plasma fibronectin were modified chemically with an ({sup 15}N,{sup 2}H)maleimide spin-label. For soluble fibronectin, both free sulfhydryl groups shown to be in confined environments as evidenced from the labeled protein exhibiting a strongly immobilized ESR spectrum as described previously using ({sup 14}N,{sup 1}H)maleimide spin-labels. When the labeled protein was adsorbed to the beads, half of the strongly immobilized component was found to convert into a weakly immobilized component, a result indicating that one of the two labeled sites becomes exposed and exhibits a fast tumbling motion. Experiments conducted using various spin-labeled fibronectin fragments suggest that the newly exposed labeled site is located between the DNA-binding and the cell-binding regions of the molecule. The data obtained indicate that, upon adsorption to polystyrene beads, plasma fibronectin undergoes a conformational change through which the buried free sulfhydryl group near the cell-binding region of the molecule is exposed. This observation may have important implications regarding the expression of cell adhesive properties of the fibronectin molecule.

  3. Covalent binding of reduced metabolites of [{sup 15}N{sub 3}]TNT to soil organic matter during a bioremediation process analyzed by {sup 15}N NMR spectroscopy

    SciTech Connect

    Achtnich, C.; Fernandes, E.; Bollag, J.M.; Knackmuss, H.J.; Lenke, H.

    1999-12-15

    Evidence is presented for the covalent binding of biologically reduced metabolites of 2,4,6-{sup 15}N{sub 3}-trinitrotoluene (TNT) to different soil fractions, using liquid {sup 15}N NMR spectroscopy. A silylation procedure was used to release soil organic matter from humin and whole soil for spectroscopic measurements. TNT-contaminated soil was spiked with 2,4,6-{sup 15}N{sub 3}-trinitrotoluene and {sup 14}C-ring labeled TNT, before treatment in a soil slurry reactor. During the anaerobic/aerobic incubation the amount of radioactivity detected in the fulvic and humic acid fractions did not change significantly whereas the radioactivity bound to humin increased to 71%. The {sup 15}N NMR spectra of the fulvic acid samples were dominated by a large peak that corresponded to aliphatic amines or ammonia. In the early stages of incubation, {sup 15}N NMR analysis of the humic acids indicated bound azoxy compounds. The signals arising from nitro and azoxy groups disappeared with further anaerobic treatment. At the end of incubation, the NMR shifts showed that nitrogen was covalently bound to humic acid as substituted amines and amides. The NMR spectra of the silylated humin suggest formation of azoxy compounds and imine linkages. Bound metabolites possessing nitro groups were also detected. Primary amines formed during the anaerobic incubation disappeared during the aerobic treatment. Simultaneously, the amount of amides and tertiary amines increased. Nitro and azoxy groups of bound molecules were still present in humin at the end of the incubation period. Formation of azoxy compounds from partially reduced TNT followed by binding and further reduction appears to be an important mechanism for the immobilization of metabolites of TNT to soil.

  4. A spin label study of egg white avidin.

    PubMed

    Chignell, C F; Starkweather, D K; Sinha, B K

    1975-07-25

    Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized

  5. 15N Content Reflects Development of Mycorrhizae and Nitrogen Dynamics During Primary Succession

    NASA Astrophysics Data System (ADS)

    Hobbie, E. A.; Jumpponen, A.

    2004-05-01

    Mycorrhizal fungi are ubiquitous symbionts on terrestrial plants that are particularly important for plant nitrogen nutrition. 15N content appears to be a useful marker of the mycorrhizal role in plant nitrogen supply because of an apparent fractionation against 15N during transfer of nitrogen from mycorrhizal fungi to host plants. Because plants developing during primary succession are gradually colonized by mycorrhizal fungi, such situations provide good opportunities to study interactions between mycorrhizal colonization and plant 15N content. Here, we present results of a study of nitrogen isotope patterns in ecosystem components during the first 100 years of ecosystem development after glacial retreat, and compare those patterns with those on adjacent mature terrain. Soils in primary succession were depleted in 15N relative to nitrogen-fixing plants. Nonmycorrhizal plants and plants generally colonized by ectomycorrhizal, ericoid, or arbuscular fungi showed similar 15N content very early in succession (-4 to -6‰ ), corresponding to low colonization levels of all plant species. Subsequent colonization of evergreen plants by ectomycorrhizal and ericoid fungi led to a 5-6‰ decline in 15N content, indicating transfer of 15N-depleted N from fungi to plants. The values recorded (-10 to -14‰ ) are among the lowest yet observed in vascular plants. Nonmycorrhizal plants and plants colonized by arbuscular mycorrhizal fungi did not decline in 15N content. Most ectomycorrhizal and saprotrophic fungi were similar in 15N content in early succession (-1 to -3‰ ), with the notable exception of ectomycorrhizal fungi suspected of proteolytic capabilities, which were 15N enriched relative to all other fungi. 15N contents in both plants and soil from the mature site were 5‰ greater than in recently exposed sites. We conclude that 1) the primary nitrogen source to this ecosystem must be atmospheric deposition, 2) low plant 15N content generally corresponds with greater

  6. Efficient production of (2)H, (13)C, (15)N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds.

    PubMed

    Zhang, Meng; Yu, Xiao-Wei; Swapna, G V T; Xiao, Rong; Zheng, Haiyan; Sha, Chong; Xu, Yan; Montelione, Gaetano T

    2016-07-13

    In order to use most modern methods of NMR spectroscopy to study protein structure and dynamics, isotope-enriched protein samples are essential. Especially for larger proteins (>20 kDa), perdeuterated and Ile (δ1), Leu, and Val methyl-protonated protein samples are required for suppressing nuclear relaxation to provide improved spectral quality, allowing key backbone and side chain resonance assignments needed for protein structure and dynamics studies. Escherichia coli and Pichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. The P. pastoris system can be used to produce (13)C, (15)N-enriched and even (2)H,(13)C, (15)N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (δ1), Leu and Val methyl-protonated groups in P. pastoris are still unavailable. Glycosylation heterogeneity also provides challenges to NMR studies. E. coli expression systems are efficient for overexpressing perdeuterated and Ile (δ1), Leu, Val methyl-protonated protein samples, but are generally not successful for producing secreted eukaryotic proteins with native disulfide bonds. The 33 kDa protein-Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using both P. pastoris and E. coli BL21 trxB (DE3) systems. Samples produced from both systems exhibit identical native disulfide bond formation and similar 2D NMR spectra, indicating similar native protein folding. The yield of (13)C, (15)N-enriched r27RCL produced using P. pastoris was 1.7 times higher that obtained using E. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced in P. pastoris exhibit O-glycosylation, while the protein samples produced in E. coli were not glycosylated. The specific activity of r27RCL from P. pastoris was ~1.4 times higher than that produced in E. coli. These data demonstrate efficient production of (2)H, (13)C, (15)N

  7. Enhanced plant growth at reduced N2O emissions: 15N dynamics confirm nitrate capture and release of co-composted biochar

    NASA Astrophysics Data System (ADS)

    Kammann, Claudia; Schmidt, Hans-Peter; Williams, Anne; Hagemann, Nikolas; Marhan, Sven; Clough, Tim; Mueller, Christoph

    2017-04-01

    Pyrogenic carbon (biochar) produced from biomass may be used as a soil amendment to sequester biomass-C and to mitigate climate change. Moreover, biochar may increase soil fertility and optimize nutrient cycling in agro-ecosystems. However, according to meta-studies large additions (>10 t ha-1) of pure biochar, may only lead to moderate yield increases. Thus, there are no economic benefits for farmers to use biochar. Recently, it has been suggested that biochar may be used as an on-farm nutrient management tool (feed additive, liquid manure treatment, composting) to deliver small doses of nutrients and biochar to the soil each year. It may also be used as a carrier for (organic) nutrients in small doses (0.5 - 2 t ha-1) in the root zone; recent studies reported considerably increased yields. The mechanisms, however, are not well understood. Co-composted biochar was recently shown to promote plant growth due to its nutrient delivery and release capabilities, particularly nitrate (NO3-). To gain further insights into biochar-nitrogen (N) interactions, we conducted a 15N labelling-tracing study under controlled conditions with treatments consisting of a non-biochar amended control, 2% (wt/wt) of untreated biochar particles (BCpure ,no intrinsic content of nitrate) and a 2% co-composted biochar (BCcomp, 5.3 g NO3-N kg-1 as a result of composting), replicated thrice. Biochars were added to a sandy loam soil in jars (200 g) planted with barley (Hordeum vulgare L.) seedlings. Fertilizer (NH4NO3) was homogenously added with either the ammonium (NH4+) or the nitrate (NO3-) pools 15N labelled (60 atom% 15N) hours before planting the seedlings. Sets of 18 jars were harvested on days 1, 3, 8, 15 and 29 and nitrogen pools were analysed to trace 15N fertilizer fate (soil mineral N, plant biomass, biochar particles, nitrous oxide (N2O) emissions). Interestingly, both BCpure and BCcomp captured fertilizer 15N from the soil matrix within hours of addition, with higher capture of 15

  8. Nitrogen stable isotope composition (δ15N) of vehicle-emitted NOx.

    PubMed

    Walters, Wendell W; Goodwin, Stanford R; Michalski, Greg

    2015-02-17

    The nitrogen stable isotope ratio of NOx (δ(15)N-NOx) has been proposed as a regional indicator for NOx source partitioning; however, knowledge of δ(15)N values from various NOx emission sources is limited. This study presents a detailed analysis of δ(15)N-NOx emitted from vehicle exhaust, the largest source of anthropogenic NOx. To accomplish this, NOx was collected from 26 different vehicles, including gasoline and diesel-powered engines, using a modification of a NOx collection method used by the United States Environmental Protection Agency, and δ(15)N-NOx was analyzed. The vehicles sampled in this study emitted δ(15)N-NOx values ranging from -19.1 to 9.8‰ that negatively correlated with the emitted NOx concentrations (8.5 to 286 ppm) and vehicle run time because of kinetic isotope fractionation effects associated with the catalytic reduction of NOx. A model for determining the mass-weighted δ(15)N-NOx from vehicle exhaust was constructed on the basis of average commute times, and the model estimates an average value of -2.5 ± 1.5‰, with slight regional variations. As technology improvements in catalytic converters reduce cold-start emissions in the future, it is likely to increase current δ(15)N-NOx values emitted from vehicles.

  9. High-Precision Measurements of 15N15N, 14N15N, and 14N2 in N2 and Potential Applications to Oceanic Nitrogen Cycle Research

    NASA Astrophysics Data System (ADS)

    Li, S.; Yeung, L.; Young, E. D.; Ostrom, N. E.; Haslun, J. A.

    2016-02-01

    The balance of nitrogen fixation and nitrogen loss in the oceans is uncertain. For example, anaerobic ammonia oxidation could account for 50% or more of marine N2 production, although its global importance is still poorly known. Isotopic ratios in fixed nitrogen species (e.g., δ15N and δ18O values of NO2- and NO3-) are widely used to trace preservation and removal of N-bearing compounds and/or isotopic variations of their different sources. However, these approaches in general probe only one side of the nitrogen mass balance—the "fixed" nitrogen reservoir—so they offer few constraints on the ultimate loss of nitrogen from that pool as N2. The rare isotopologue ratio 15N15N/14N2 in N2may provide information about those nitrogen-loss processes directly. We will report the first measurements of Δ30 (the abundance of 15N15N relative to that predicted by chance alone), made on a unique high-resolution mass spectrometer (the Nu Instruments Panorama), and we will discuss the potential utility of Δ30 as an independent tracer of the nitrogen cycle. The parameter Δ30 is insensitive to the bulk 15N/14N isotopic ratio of the reservoir; instead, it reflects isotopic ordering in N2, which is altered when N-N bonds are made or broken. Our preliminary measurements of N2 from denitrifying soils and pure cultures of denitrifiers indicate large kinetic isotopic effects during N-N bond formation that favor 15N15N production during denitrification. We also observed a nonstochastic excess of 15N15N in tropospheric N2 [Δ30 = +19.05 ± 0.12‰ (1σ)]. This excess likely comes from fixed-nitrogen loss processes in the biosphere. Variations in Δ30 of N2 from pure culture experiments (+16.96 to +18.95‰) probably reflect the different isotopic signatures of the enzymes that catalyze denitrification. So, enzyme-specific Δ30 values of dissolved N2 should provide information about the importance of different biochemical pathways of fixed-nitrogen loss (e.g., denitrification vs

  10. An evaluation of sources of nitrogen in shallow groundwater using (15)N abundance technique.

    PubMed

    Alva, A K; Dou, H; Paramasivam, S; Wang, F L; Graetz, D A; Sajwan, K S

    2006-01-01

    A (15)N abundance technique was employed to identify the source of NO(3)-N in groundwater under three commercial citrus production sites in central Florida. Water samples were collected from 0 to 300 and 300 to 600 cm depths in the surficial aquifer and analyzed for NO(3)-N and delta N-15 (delta (15)N). Groundwater samples were also collected in a residential area adjacent to one of the citrus groves and analyzed for NO(3)-N and delta (15)N. The delta (15)N values were in the range of (+)1 to (+)10% in both depths underneath the citrus groves. The range of delta (15)N measured in this study represents the range expected for groundwater that was impacted by NO(3)-N originated from mineralization of organic N from the soil as well as from the crop residue. There are occasional high delta (15)N values which are indicative of the effects of NH(3) volatilization losses of applied fertilizer N. The range of delta (15)N values for groundwater samples collected from the residential area adjacent to the citrus groves was very similar to that from the groundwater underneath the citrus groves. Thus, the source of NO(3)-N that impacted the groundwater under the citrus groves also impacted the groundwater in the adjacent residential area.

  11. Entanglement of Spin States in 15N@C60

    NASA Astrophysics Data System (ADS)

    Scherer, W.; Weidinger, A.; Mehring, M.

    2004-09-01

    The endohedral fullerene 15N@C60 comprises an electron spin S = 3/2 coupled to a nuclear spin I = 1/2 and is therefore ideally suited for experimental testing of basic properties of quantum mechanics. We will show that the 15N@C60 molecule represents a multi qubit system where different kinds of entangled states can be generated.

  12. Nitrate reduction in a groundwater microcosm determined by sup 15 N gas chromatography-mass spectrometry

    SciTech Connect

    Bengtsson, G.; Annadotter, H. )

    1989-11-01

    Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO{sub 3}{sup {minus}}-N liter{sup {minus}1}. Traces of {sup 15}NO{sub 3}{sup {minus}} were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day{sup {minus}1}. Both assimilatory and dissimilatory reduction rates were estimated from analyses of {sup 15}N{sub 2}, {sup 15}N{sub 2}O, {sup 15}NH{sub 4}{sup +}, and {sup 15}N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N{sub 2} and N{sub 2}O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO{sub 3}{sup {minus}} and NH{sub 4}{sup +} were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their (methyl-{sup 3}H)thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g{sup {minus}1} day{sup {minus}1}. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N{sub 2}, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO{sub 3}{sup {minus}} was reduced to NH{sub 4}{sup +}. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month.

  13. Chemo-enzymatic synthesis of selectively ¹³C/¹⁵N-labeled RNA for NMR structural and dynamics studies.

    PubMed

    Alvarado, Luigi J; Longhini, Andrew P; LeBlanc, Regan M; Chen, Bin; Kreutz, Christoph; Dayie, T Kwaku

    2014-01-01

    RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (~25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific (13)C/(15)N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling, and selective-biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example, total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10%) that drop precipitously for oligonucleotides larger than 50 nt. The alternative method of de novo pyrimidine biosynthesis of NTPs is also a robust technique, with modest yields of up to 45%, but it comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1' and C5' and the pyrimidine nucleobase C2, C4, C5, or C6. Biomass-produced, uniformly or selectively labeled NTPs offer a third method, but suffer from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of (13)C-(13)C couplings. In contrast to these four methods, our current chemo-enzymatic approach overcomes most of these shortcomings and allows

  14. Your emotion or mine: labeling feelings alters emotional face perception—an ERP study on automatic and intentional affect labeling

    PubMed Central

    Herbert, Cornelia; Sfärlea, Anca; Blumenthal, Terry

    2013-01-01

    Empirical evidence suggests that words are powerful regulators of emotion processing. Although a number of studies have used words as contextual cues for emotion processing, the role of what is being labeled by the words (i.e., one's own emotion as compared to the emotion expressed by the sender) is poorly understood. The present study reports results from two experiments which used ERP methodology to evaluate the impact of emotional faces and self- vs. sender-related emotional pronoun-noun pairs (e.g., my fear vs. his fear) as cues for emotional face processing. The influence of self- and sender-related cues on the processing of fearful, angry and happy faces was investigated in two contexts: an automatic (experiment 1) and intentional affect labeling task (experiment 2), along with control conditions of passive face processing. ERP patterns varied as a function of the label's reference (self vs. sender) and the intentionality of the labeling task (experiment 1 vs. experiment 2). In experiment 1, self-related labels increased the motivational relevance of the emotional faces in the time-window of the EPN component. Processing of sender-related labels improved emotion recognition specifically for fearful faces in the N170 time-window. Spontaneous processing of affective labels modulated later stages of face processing as well. Amplitudes of the late positive potential (LPP) were reduced for fearful, happy, and angry faces relative to the control condition of passive viewing. During intentional regulation (experiment 2) amplitudes of the LPP were enhanced for emotional faces when subjects used the self-related emotion labels to label their own emotion during face processing, and they rated the faces as higher in arousal than the emotional faces that had been presented in the “label sender's emotion” condition or the passive viewing condition. The present results argue in favor of a differentiated view of language-as-context for emotion processing. PMID:23888134

  15. Availability of 15N from pioneer herbaceous plants to pine seedlings in reclaimed burnt soils.

    PubMed

    González-Prieto, S J; Villar, M C; Carballas, T

    2008-09-01

    A pot experiment was used to assess N uptake by pine seedlings during 2 years on a burnt soil to which was added (15)N-labelled ryegrass, obtained from a (15)N-enriched sample of this soil after a fire. The nitrogen concentration in needles, stems and roots of seedlings decreased significantly from the first to the second growing period (from 2.55, 1.30 and 2.19% to 1.19, 0.47 and 1.00%, respectively), with needles accounting for 53-58% of the pine-N. At the end of the experiment, 98.87 +/- 1.12% of the added ryegrass-(15)N was recovered: two-thirds in the soil organic N pool and one-third in the pine seedlings. Therefore, the post-fire pulse of inorganic-N, which was successfully kept in the burnt soil-plant system through its uptake by the pioneer species, is available for trees in the medium term. Pine seedlings assimilated 16.4% and 16.9% of the added ryegrass-(15)N in the first and second year, respectively. This result contrasts with the usual yearly decrease of added N uptake by plants; a possible explanation is the transient increase of available N in burnt soils that would have modified the mineralization pattern of the (15)N-labelled phytomass. The pine-N derived from the ryegrass-N decreased from 4.05% in the first year to 2.53% in the second one, with 3.10% being the 2-year weighed average. In addition to the direct contribution of ryegrass to pine-N nutrition reflected by these figures, the rapid post-fire establishment of a herbaceous cover on the burnt soil also provides important indirect benefits for tree nutrition by reducing organic- and inorganic-N losses. Copyright (c) 2008 John Wiley & Sons, Ltd.

  16. Light-mediated 15N fractionation in Caribbean gorgonian octocorals: implications for pollution monitoring

    NASA Astrophysics Data System (ADS)

    Baker, D. M.; Kim, K.; Andras, J. P.; Sparks, J. P.

    2011-09-01

    The stable nitrogen isotope ratio ( δ 15N) of coral tissue is a useful recorder of anthropogenic pollution in tropical marine ecosystems. However, little is known of the natural environmentally induced fractionations that affect our interpretation of coral δ 15N values. In symbiotic scleractinians, light affects metabolic fractionation of N during photosynthesis, which may confound the identification of N pollution between sites of varied depth or turbidity. Given the superiority of octocorals for δ 15N studies, our goal was to quantify the effect of light on gorgonian δ 15N in the context of monitoring N pollution sources. Using field collections, we show that δ 15N declined by 1.4‰ over 20 m depth in two species of gorgonians, the common sea fan, Gorgonia ventalina, and the slimy sea plume, Pseudopterogorgia americana. An 8-week laboratory experiment with P. americana showed that light, not temperature causes this variation, whereby the lowest fractionation of the N source was observed in the highest light treatment. Finally, we used a yearlong reciprocal depth transplant experiment to quantify the time frame over which δ 15N changes in G. ventalina as a function of light regime . Over the year, δ 15N was unchanged and increased slightly in the deep control colonies and shallow colonies transplanted to the deep site, respectively. Within 6 months, colonies transplanted from deep to shallow became enriched by 0.8‰, mirroring the enrichment observed in the shallow controls, which was likely due to the combined effect of an increase in the source δ 15N and reduced fractionation. We conclude that light affects gorgonian δ 15N fractionation and should be considered in sampling designs for N pollution monitoring. However, these fractionations are small relative to differences observed between natural and anthropogenic N sources.

  17. Foliar retention of 15N-nitrate and 15N-ammonium by red maple (Acer rubrum) and white oak (Quercus alba) leaves from simulated rain

    SciTech Connect

    Garten Jr, Charles T; Hanson, Paul J

    1990-07-01

    Studies of nitrogen cycling in forests indicate that trees assimilate atmospheric nitrate and ammonium and that differences between atmospheric deposition to the forest canopy and deposition measured in forest throughfall can be attributed to the removal of these ions from rain by tree leaves. Red maple and white oak leaves were exposed to artificial rain solutions (pH 4.1) containing {sup 15}N-labeled nitrate (3.5 {micro}g N/ml) or ammonium (2.2 {micro}g N/ml). At two time intervals after exposure (2 hr and 2 days) an exposed leaf and a control (non-exposed) leaf were removed from replicate seedlings. Based on results from {sup 15}N analysis, most of the nitrate applied to tree leaves was removed by washing with water; the mean per cent removal ({+-} standard error, N = 4) was 87 {+-} 1 and 73 {+-} 4% of the {sup 15}NO-N Applied to red maple and white oak leaves, respectively. Relative retention of {sup 15}NH{sub 4}-N by the leaves was greater than that observed for {sup 15}NO{sub 3}-N. In red maple and white oak leaves, 58 {+-} 9 and 84 {+-} 7% (mean {+-} standard error, N = 4), respectively, of the applied ammonium was not removed by washing treatments. Our results show that the foliar uptake of {sup 15}NH{sub 4}{sup +} from simulated rain by deciduous tree leaves is greater than that for {sup 15}NO{sub 3}{sup -}. Greater retention of NH{sub 4}{sup +} than NO{sub 3}{sup -} ions by red maple and white oak leaves from simulated rainfall is consistent with field observations showing a preferential retention of ammonium from rainfall by forest canopies. As nitrogen chemistry and the relative importance of nitrogen compounds in the atmosphere change in response to proposed emission reductions (and possibly climate change), an improved understanding of the fate of airborne nitrogen compounds in forest biogeochemical cycles will be necessary.

  18. Study of E/Z isomerization of (arylamino)methylidenefuran-2(3H)-ones by (1) H, (13) C, (15) N spectroscopy and DFT calculations in different solvents.

    PubMed

    Osipov, Alexander K; Anis'kov, Alexander A; Grinev, Vyacheslav S; Yegorova, Alevtina Yu

    2017-08-01

    The structure and configuration of the series of previously unknown arylaminomethylidenefuran-2(3H)-ones have been determined in solution by (1) H, (13) C, (15) N nuclear magnetic resonance spectroscopy including two-dimensional experiments such as (1) H─(1) H COSY, dqCOSY, (1) H─(13) C HSQC, (1) H─(13) C HMBC. It was found that synthesized substances exist as an equilibrium mixture of E- and Z-enamines in solution. It was established on the basis of density functional theory calculations that the exchange between the two push-pull enamines is a simple rotation around an exocyclic partial double bond that depends on the effect of the solvents. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  19. Synthesis of 14C-Labelled Octahydor-1,3,5,7-Tetranitro-1,3,5,7-Tetrazocine (HMx0 and 15N-Isotopic Hexahyrro-1,3,5-Trinitro-1,3,5-Triazine (RDX) for use in Microcosm Experiments.

    DTIC Science & Technology

    2000-02-01

    bioremediation process. To synthesize C(14)HMX, acetylation of labelled hexamethylenetetramine (C(14)HMTA) was done yielding 3,7-diacetyl-1,3,5,7... hexamethylenetetramine (N(15)HMTA) was done according to the Hale Process. N(15)HMTA was prepared by reaching cold formaldehyde with isotopic nitrogen-15 ammonium hydroxide.

  20. Estimate of production of gaseous nitrogen in the human body based on (15)N analysis of breath N2 after administration of [(15)N2]urea.

    PubMed

    Junghans, Peter

    2013-01-01

    After oral administration of [(15)N2]urea (1.5 mmol, 95 atom% (15)N), we found that breath N2 was significantly (15)N-labelled. The result suggests that molecular nitrogen in breath must be partly produced endogenously. Based on a metabolic model, the endogenous N2 production was estimated to be 0.40±0.25 mmol kg(-1) d(-1) or 2.9±1.8 % of the total (urinary and faecal) N excretion in fasted healthy subjects (n=4). In patients infected with Helicobacter pylori (n=5), the endogenous N2 production was increased to 1.24±0.59 mmol kg(-1) d(-1) or 9.0±4.3 % of the total N excretion compared to the healthy controls (p<0.05). We conclude that N balance and gas exchange measurements may be affected by endogenously produced nitrogen, especially in metabolic situations with elevated nitrosation, for instance in oxidative and nitrosative stress-related diseases such as H. pylori infections.

  1. Proton-coupled 15N NMR spectra of neutral and protonated ethenoadenosine and ethenocytidine.

    PubMed Central

    Sierzputowska-Gracz, H; Wiewiórowski, M; Kozerski, L; von Philipsborn, W

    1984-01-01

    The 15N chemical shifts and 15N, 1H spin coupling constants were determined in the title compounds using the INEPT pulse sequence and assigned with the aid of selective proton decoupling. The delta/15N/ and J/N, H/ values are discussed in terms of involvement of the imidazole ring created by ethenobridging in the electronic structure of the whole molecule. Both spectral parameters indicate that the diligant nitrogen in this ring is the primary site of protonation in these modified nucleosides. It is concluded that 15N NMR of nucleoside bases can be largely a complementary method to 1H and 13C NMR studies and, in addition, can serve as a direct probe for studies of nitrogen environment in oligomeric fragments of nucleic acids even at moderately strong magnetic fields due to the higher spectral dispersion compared with 1H and 13C NMR spectra. PMID:6473107

  2. Exploring Whiteness: A Study of Self Labels for White Americans.

    ERIC Educational Resources Information Center

    Martin, Judith N.; Krizek, Robert L.; Nakayama, Thomas K.; Bradford, Lisa

    1996-01-01

    Examines the preferences and meanings of labels for White Americans as discursively defined expressions of identity, after preliminary revelations of resistance by Whites to self-labeling was seen. Surveys 371 White undergraduate students, rating seven labels regarding preference and discussing feelings about self-labeling. Reveals that the most…

  3. Exploring Whiteness: A Study of Self Labels for White Americans.

    ERIC Educational Resources Information Center

    Martin, Judith N.; Krizek, Robert L.; Nakayama, Thomas K.; Bradford, Lisa

    1996-01-01

    Examines the preferences and meanings of labels for White Americans as discursively defined expressions of identity, after preliminary revelations of resistance by Whites to self-labeling was seen. Surveys 371 White undergraduate students, rating seven labels regarding preference and discussing feelings about self-labeling. Reveals that the most…

  4. Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.

    PubMed

    Fan, Ying; Shi, Lichi; Ladizhansky, Vladimir; Brown, Leonid S

    2011-02-01

    Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly (13)C,(15)N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution (13)C and (15)N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

  5. Nitrogen source tracking with δ15N content of coastal wetland plants in Hawaii

    Treesearch

    Gregory L. Bruland; Richard A.. Mackenzie

    2010-01-01

    Inter- and intra-site comparisons of the nitrogen (N) stable isotope composition of wetland plant species have been used to identify sources of N in coastal areas. In this study, we compared δ15N values from different herbaceous wetland plants across 34 different coastal wetlands from the five main Hawaiian Islands and investigated relationships of δ15N with...

  6. MASS LOSS AND NITROGEN DYNAMICS DURING THE DECOMPOSITION OF A N-LABELED N2-FIXING EPOPHYTIC LICHEN, LOBARIA OREGANA (TUCK.) MULL. ARG.

    EPA Science Inventory

    We studied mass loss and nitrogen dynamics during fall and spring initiated decomposition of an N2-fixing epiphytic lichen, Lobaria oregana (Tuck.) Mull. Arg. using 15N. We developed a method of labeling lichens with 15N that involved spraying lichen material with a nutrient sol...

  7. MASS LOSS AND NITROGEN DYNAMICS DURING THE DECOMPOSITION OF A N-LABELED N2-FIXING EPOPHYTIC LICHEN, LOBARIA OREGANA (TUCK.) MULL. ARG.

    EPA Science Inventory

    We studied mass loss and nitrogen dynamics during fall and spring initiated decomposition of an N2-fixing epiphytic lichen, Lobaria oregana (Tuck.) Mull. Arg. using 15N. We developed a method of labeling lichens with 15N that involved spraying lichen material with a nutrient sol...

  8. Compound-Specific δ15N Amino Acid Measurements in Littoral Mussels in the California Upwelling Ecosystem: A New Approach to Generating Baseline δ15N Isoscapes for Coastal Ecosystems

    PubMed Central

    Vokhshoori, Natasha L.; McCarthy, Matthew D.

    2014-01-01

    We explored δ15N compound-specific amino acid isotope data (CSI-AA) in filter-feeding intertidal mussels (Mytilus californianus) as a new approach to construct integrated isoscapes of coastal primary production. We examined spatial δ15N gradients in the California Upwelling Ecosystem (CUE), determining bulk δ15N values of mussel tissue from 28 sites between Port Orford, Oregon and La Jolla, California, and applying CSI-AA at selected sites to decouple trophic effects from isotopic values at the base of the food web. Bulk δ15N values showed a strong linear trend with latitude, increasing from North to South (from ∼7‰ to ∼12‰, R2 = 0.759). In contrast, CSI-AA trophic position estimates showed no correlation with latitude. The δ15N trend is therefore most consistent with a baseline δ15N gradient, likely due to the mixing of two source waters: low δ15N nitrate from the southward flowing surface California Current, and the northward transport of the California Undercurrent (CUC), with15N-enriched nitrate. This interpretation is strongly supported by a similar linear gradient in δ15N values of phenylalanine (δ15NPhe), the best AA proxy for baseline δ15N values. We hypothesize δ15NPhe values in intertidal mussels can approximate annual integrated δ15N values of coastal phytoplankton primary production. We therefore used δ15NPhe values to generate the first compound-specific nitrogen isoscape for the coastal Northeast Pacific, which indicates a remarkably linear gradient in coastal primary production δ15N values. We propose that δ15NPhe isoscapes derived from filter feeders can directly characterize baseline δ15N values across major biochemical provinces, with potential applications for understanding migratory and feeding patterns of top predators, monitoring effects of climate change, and study of paleo- archives. PMID:24887109

  9. Spin labeled acetylcholine analogs: studies of cholinergic receptor.

    PubMed

    Rosen, G M; Abou-Donia, M B; Yeh, J Z; Menzel, D B

    1975-10-01

    Some spin-labeled acetylcholine analogs, in which the number of methylene groups between the quaternary nitrogen and the ether oxygen ranged between 1-5, were synthesized to study drug interacitons with acetylcholine receptors. None of the compounds tested, with the exception of the one that contained 2 methylene groups (SL-2) had any cholinergic activity. SL-2 was not capable of producing any nicotinic cholinomimetic activity. On the other hand it proved to have a very weak nicotinic cholinolytic activity on the receptors of the frog satorius muscle. This compound exhibited strong antagonism against muscarinic receptors of the isolated frog heart. The muscarinic cholinolytic action of the spin-label ACh analog is discussed in terms of the molecular perturbation theory of drug action.

  10. Bromodeoxyuridine-labeled oligonucleotides as tools for oligonucleotide uptake studies.

    PubMed

    Maszewska, Maria; Kobylańska, Anna; Gendaszewska-Darmach, Edyta; Koziołkiewicz, Maria

    2002-12-01

    The mechanisms by which various oligonucleotides (ODNs) and their analogs enter cells are not fully understood. A common technique used in studies on cellular uptake of ODNs is their conjugation with fluorochromes. However, fluorescently labeled ODNs may vary from the parent compounds in charge and hydrophilicity, and they may interact differently with some components of cellular membranes. In this report, we present an alternative method based on the immunofluorescent detection of ODNs with incorporated 5-bromo-2'-deoxyuridine (BrdUrd). Localization of BrdUrd-modified ODNs has been achieved using FITC-labeled anti-BrdUrd antibodies. This technique allowed determination of the differences in cellular uptake of phosphodiester (PO) and phosphorothioate (PS) ODNs and their derivatives conjugated with cholesterol and menthol. The immunocytochemical method also has shown that the cellular uptake of some ODNs may be influenced by specific sequences that are responsible for the formation of higher-order structures.

  11. Eastern oyster (Crassostrea virginica) δ15N as a bioindicator of nitrogen sources: Observations and modeling

    PubMed Central

    Fertig, B.; Carruthers, T.J.B.; Dennison, W.C.; Fertig, E.J.; Altabet, M.A.

    2013-01-01

    Stable nitrogen isotopes (δ15N) in bioindicators are increasingly employed to identify nitrogen sources in many ecosystems and biological characteristics of the eastern oyster (Crassostrea virginica) make it an appropriate species for this purpose. To assess nitrogen isotopic fractionation associated with assimilation and baseline variations in oyster mantle, gill, and muscle tissue δ15N, manipulative fieldwork in Chesapeake Bay and corresponding modeling exercises were conducted. This study (1) determined that five individuals represented an optimal sample size; (2) verified that δ15N in oysters from two locations converged after shared deployment to a new location reflecting a change in nitrogen sources; (3) identified required exposure time and temporal integration (four months for muscle, two to three months for gill and mantle); and (4) demonstrated seasonal δ15N increases in seston (summer) and oysters (winter). As bioindicators, oysters can be deployed for spatial interpolation of nitrogen sources, even in areas lacking extant populations. PMID:20381097

  12. Thulium-170-labeled microparticles for local radiotherapy: preliminary studies.

    PubMed

    Polyak, Andras; Das, Tapas; Chakraborty, Sudipta; Kiraly, Reka; Dabasi, Gabriella; Joba, Robert Peter; Jakab, Csaba; Thuroczy, Julianna; Postenyi, Zita; Haasz, Veronika; Janoki, Gergely; Janoki, Gyozo A; Pillai, Maroor R A; Balogh, Lajos

    2014-10-01

    The present article describes the preparation, characterization, and biological evaluation of Thulium-170 ((170)Tm) [T1/2 = 128.4 days; Eβmax = 968 keV; Eγ = 84 keV (3.26%)] labeled tin oxide microparticles for its possible use in radiation synovectomy (RSV) of medium-sized joints. (170)Tm was produced by irradiation of natural thulium oxide target. 170Tm-labeled microparticles were synthesized with high yield and radionuclidic purity (> 99%) along with excellent in vitro stability by following a simple process. Particle sizes and morphology of the radiolabeled particles were examined by light microscope, dynamic light scattering, and transmission electron microscope and found to be of stable spherical morphology within the range of 1.4-3.2 μm. The preparation was injected into the knee joints of healthy Beagle dogs intraarticularly for biological studies. Serial whole-body and regional images were taken by single-photon-emission computed tomography (SPECT) and SPECT-CT cameras up to 9 months postadministration, which showed very low leakage (< 8% of I.D.) of the instilled particles. The majority of leaked radiocolloid particles were found in inguinal lymph nodes during the 9 months of follow-up. All the animals tolerated the treatment well; the compound did not show any possible radiotoxicological effect. These preliminary studies showed that 170Tm-labeled microparticles could be a promising nontoxic and effective radiopharmaceutical for RSV applications or later local antitumor therapy.

  13. Chick embryo proliferation studies using EdU labeling.

    PubMed

    Warren, Michelle; Puskarczyk, Karolina; Chapman, Susan C

    2009-04-01

    Cell proliferation studies are an important experimental tool. The most commonly used thymidine analogues, tritiated thymidine and bromodeoxyuridine (BrdU) label cells during S-phase. Both methods have significant drawbacks: low sensitivity in the case of tritiated thymidine and a denaturation step during BrdU detection that destroys most cellular epitopes, requiring careful optimization. The antibody against BrdU is also large and tissue penetration can be difficult. EdU (5'-ethynyl-2'-deoxyuridine) is closely chemically related to BrdU, with detection achieved by a copper catalyzed reaction requiring a small fluorescently conjugated azide. Cell cultures, flow cytometry and high throughput studies using EdU-labeled cells is exceptionally fast and does not require denaturation or antibodies. We have developed a tissue-labeling technique in chick embryos using EdU. Following EdU chemistry to detect proliferating cells, the tissue can undergo immunolabeling. We demonstrate fluorescent EdU chemistry followed by Tuj1 antibody staining resulting in multiplex fluorescent tissues.

  14. The first synthesis of [9,Amino-{sup 15}N{sub 2}]adenine and {beta}- 2{prime}-Deoxy-[9,Amino-{sup 15}N{sub 2}]adenosine

    SciTech Connect

    Orji, C.C.; Silks, L.A. III

    1995-12-31

    {beta}-2{prime}-Deoxy-[9, Amino-{sup 15}N{sub 2}] Adenosine has been constructed in 4 steps from commercially available 5-amino-4,6-dichloropyrimidine and {sup 15}NH{sub 3}. The reactions have been scaled provide grams quantities of labeled nucleoside.

  15. Metamorphic origin of ore-forming fluids for orogenic gold-bearing quartz vein systems in the North American Cordillera: constraints from a reconnaissance study of δ15N, δD, and δ18O

    USGS Publications Warehouse

    Jia, Y.; Kerrich, R.; Goldfarb, R.

    2003-01-01

    The western North American Cordillera hosts a large number of gold-bearing quartz vein systems from the Mother Lode of southern California, through counterparts in British Columbia and southeastern Alaska, to the Klondike district in central Yukon. These vein systems are structurally controlled by major fault zones, which are often reactivated terrane-bounding sutures that formed in orogens built during accretion and subduction of terranes along the continental margin of North America. Mineralization ages span mid-Jurassic to early Tertiary and encompass much of the evolution ofthe Cordilleran orogen. Nitrogen contents and δ15N values of hydrothermal micas from veins are between 130 and 3,500 ppm and 1.7 to 5.5 per mil, respectively. These values are consistent with fluids derived from metamorphic dehydration reactions within the Phanerozoic accretion-subduction complexes, which have δ15N values of 1 to 6 per mil. The δ18O values of gold-bearing vein quartz from different locations in the Cordillera are between 14.6 and 22.2 per mil but are uniform for individual vein systems. The δD values of hydrothermal micas are between -110 and -60 per mil. Ore fluids have calculated δ18O values of 8 to 16 per mil and δD values of -65 to -10 per mil at an estimated temperature of 300δC; δD values of ore fluids do not show any latitudinal control. These results indicate a deep crustal source for the ore-forming fluids, most likely of metamorphic origin. Low δDH2O values of -120 to -130 per mil for a hydrous muscovite from the Sheba vein in the Klondike district reflect secondary exchange between recrystallizing mica and meteoric waters. Collectively, the N, H, and O isotope compositions of ore-related hydrothermal minerals indicate that the formation of these gold-bearing veins involved dilute, aqueous carbonic, and nitrogen-bearing fluids that were generated from metamorphic dehydration reactions at deep crustal levels. These data are not consistent with either mantle

  16. Influence of open ocean nitrogen supply on the skeletal δ15N of modern shallow-water scleractinian corals

    NASA Astrophysics Data System (ADS)

    Wang, Xingchen T.; Sigman, Daniel M.; Cohen, Anne L.; Sinclair, Daniel J.; Sherrell, Robert M.; Cobb, Kim M.; Erler, Dirk V.; Stolarski, Jarosław; Kitahara, Marcelo V.; Ren, Haojia

    2016-05-01

    The isotopic composition of skeleton-bound organic nitrogen in shallow-water scleractinian corals (hereafter, CS-δ15N) is an emerging tool for studying the marine nitrogen cycle in the past. The CS-δ15N has been shown to reflect the δ15N of nitrogen (N) sources to corals, with most applications to date focusing on the anthropogenic/terrestrial N inputs to reef environments. However, many coral reefs receive their primary N sources from the open ocean, and the CS-δ15N of these corals may provide information on past changes in the open ocean regional and global N cycle. Using a recently developed persulfate/denitrifier-based method, we measured CS-δ15N in modern shallow-water scleractinian corals from 8 sites proximal to the open ocean. At sites with low open ocean surface nitrate concentrations typical of the subtropics and tropics, measured CS-δ15N variation on seasonal and annual timescales is most often less than 2‰. In contrast, a broad range in CS-δ15N (of ∼10‰) is measured across these sites, with a strong correlation between CS-δ15N and the δ15N of the deep nitrate supply to the surface waters near the reefs. While CS-δ15N can be affected by other N sources as well and can vary in response to local reef conditions as well as coral/symbiont physiological changes, this survey indicates that, when considering corals proximal to the open ocean, the δ15N of the subsurface nitrate supply to surface waters drives most of the CS-δ15N variation across the global ocean. Thus, CS-δ15N is a promising proxy for reconstructing the open ocean N cycle in the past.

  17. A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation.

    PubMed

    Desclos, Marie; Etienne, Philippe; Coquet, Laurent; Jouenne, Thierry; Bonnefoy, Josette; Segura, Raphaël; Reze, Sandrine; Ourry, Alain; Avice, Jean-Christophe

    2009-07-01

    Our goal was to identify the leaf proteomic changes which appeared during N remobilisation that were associated or not associated with senescence of oilseed rape in response to contrasting nitrate availability. Remobilisation of N and leaf senescence status were followed using (15)N tracing, patterns of chlorophyll level, total protein content and a molecular indicator based on expression of senescence-associated gene 12/Cab genes. Three phases associated with N remobilisation were distinguished. Proteomics revealed that 55 proteins involved in metabolism, energy, detoxification, stress response, proteolysis and protein folding, were significantly induced during N remobilisation. Four proteases were specifically identified. FtsH, a chloroplastic protease, was induced transiently during the early stages of N remobilisation. Considering the dynamics of N remobilisation, chlorophyll and protein content, the pattern of FtsH expression indicated that this protease could be involved in the degradation of chloroplastic proteins. Aspartic protease increased at the beginning of senescence and was maintained at a high level, implicating this protease in proteolysis during the course of leaf senescence. Two proteases, proteasome beta subunit A1 and senescence-associated gene 12, were induced and continued to increase during the later phase of senescence, suggesting that these proteases are more specifically involved in the proteolysis processes occurring at the final stages of leaf senescence.

  18. Ecosystem N distribution and δ15N during a century of forest regrowth after agricultural abandonment

    USGS Publications Warehouse

    Compton, J.E.; Hooker, T.D.; Perakis, S.S.

    2007-01-01

    Stable isotope ratios of terrestrial ecosystem nitrogen (N) pools reflect internal processes and input–output balances. Disturbance generally increases N cycling and loss, yet few studies have examined ecosystem δ15N over a disturbance-recovery sequence. We used a chronosequence approach to examine N distribution and δ15N during forest regrowth after agricultural abandonment. Site ages ranged from 10 to 115 years, with similar soils, climate, land-use history, and overstory vegetation (white pine Pinus strobus). Foliar N and δ15N decreased as stands aged, consistent with a progressive tightening of the N cycle during forest regrowth on agricultural lands. Over time, foliar δ15N became more negative, indicating increased fractionation along the mineralization–mycorrhizal–plant uptake pathway. Total ecosystem N was constant across the chronosequence, but substantial internal N redistribution occurred from the mineral soil to plants and litter over 115 years (>25% of ecosystem N or 1,610 kg ha−1). Temporal trends in soil δ15N generally reflected a redistribution of depleted N from the mineral soil to the developing O horizon. Although plants and soil δ15N are coupled over millennial time scales of ecosystem development, our observed divergence between plants and soil suggests that they can be uncoupled during the disturbance-regrowth sequence. The approximate 2‰ decrease in ecosystem δ15N over the century scale suggests significant incorporation of atmospheric N, which was not detected by traditional ecosystem N accounting. Consideration of temporal trends and disturbance legacies can improve our understanding of the influence of broader factors such as climate or N deposition on ecosystem N balances and δ15N.

  19. 15N NMR of 1,4-dihydropyridine derivatives.

    PubMed

    Goba, Inguna; Liepinsh, Edvards

    2013-07-01

    In this article, we describe the characteristic (15)N and (1)HN NMR chemical shifts and (1)J((15)N-(1)H) coupling constants of various symmetrically and unsymmetrically substituted 1,4-dihydropyridine derivatives. The NMR chemical shifts and coupling constants are discussed in terms of their relationship to structural features such as character and position of the substituent in heterocycle, N-alkyl substitution, nitrogen lone pair delocalization within the conjugated system, and steric effects.

  20. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility.

    PubMed

    Kobayashi, Hiroshi; Swapna, G V T; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T; Inouye, Masayori

    2012-04-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS(2)) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS(2)-tag is replaced with non-isotope labeled PrS(2)-tag, silencing the NMR signals from PrS(2)-tag in isotope-filtered (1)H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS(2)-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS(2) (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS(2)-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS(2)-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone (1)H, (15)N and (13)C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear (1)H-(15)N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

  1. δ15N Value Does Not Reflect Fasting in Mysticetes

    PubMed Central

    Aguilar, Alex; Giménez, Joan; Gómez–Campos, Encarna; Cardona, Luís; Borrell, Asunción

    2014-01-01

    The finding that tissue δ15N values increase with protein catabolism has led researchers to apply this value to gauge nutritive condition in vertebrates. However, its application to marine mammals has in most occasions failed. We investigated the relationship between δ15N values and the fattening/fasting cycle in a model species, the fin whale, a migratory capital breeder that experiences severe seasonal variation in body condition. We analyzed two tissues providing complementary insights: one with isotopic turnover (muscle) and one that keeps a permanent record of variations in isotopic values (baleen plates). In both tissues δ15N values increased with intensive feeding but decreased with fasting, thus contradicting the pattern previously anticipated. The apparent inconsistency during fasting is explained by the fact that a) individuals migrate between different isotopic isoscapes, b) starvation may not trigger significant negative nitrogen balance, and c) excretion drops and elimination of 15N-depleted urine is minimized. Conversely, when intensive feeding is resumed in the northern grounds, protein anabolism and excretion start again, triggering 15N enrichment. It can be concluded that in whales and other mammals that accrue massive depots of lipids as energetic reserves and which have limited access to drinking water, the δ15N value is not affected by fasting and therefore cannot be used as an indicatior of nutritive condition. PMID:24651388

  2. Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda

    PubMed Central

    Di Paolo, Alexandre; Duval, Valérie; Matagne, André

    2010-01-01

    Lysozyme from lambda bacteriophage (λ lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, λ lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of λ lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes λ lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of λ lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the 1H, 13C and 15N backbone resonance assignments for λ lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR. PMID:20300891

  3. Alkaline Hydrolysis/Polymerization of 2,4,6-Trinitrotoluene: Characterization of Products by 13C and 15N NMR

    USGS Publications Warehouse

    Thorn, K.A.; Thorne, P.G.; Cox, L.G.

    2004-01-01

    Alkaline hydrolysis has been investigated as a nonbiological procedure for the destruction of 2,4,6-trinitrotoluene (TNT) in explosives contaminated soils and munitions scrap. Nucleophilic substitutions of the nitro and methyl groups of TNT by hydroxide ion are the initial steps in the alkaline degradation of TNT. Potential applications of the technique include both in situ surface liming and ex situ alkaline treatment of contaminated soils. A number of laboratory studies have reported the formation of an uncharacterized polymeric material upon prolonged treatment of TNT in base. As part of an overall assessment of alkaline hydrolysis as a remediation technique, and to gain a better understanding of the chemical reactions underlying the hydrolysis/polymerization process, the soluble and precipitate fractions of polymeric material produced from the calcium hydroxide hydrolysis of unlabeled and 15N-labeled TNT were analyzed by elemental analysis and 13C and 15N nuclear magnetic resonance spectroscopy. Spectra indicated that reactions leading to polymerization included nucleophilic displacement of nitro groups by hydroxide ion, formation of ketone, carboxyl, alcohol, ether, and other aliphatic carbons, conversion of methyl groups to diphenyl methylene carbons, and recondensation of aromatic amines and reduced forms of nitrite, including ammonia and possibly hydroxylamine, into the polymer. Compared to the distribution of carbons in TNT as 14% sp 3- and 86% sp2-hybridized, the precipitate fraction from hydrolysis of unlabeled TNT contained 33% sp3- and 67% sp 2-hybridized carbons. The concentration of nitrogen in the precipitate was 64% of that in TNT. The 15N NMR spectra showed that, in addition to residual nitro groups, forms of nitrogen present in the filtrate and precipitate fractions include aminohydroquinone, primary amide, indole, imine, and azoxy, among others. Unreacted nitrite was recovered in the filtrate fraction. The toxicities and susceptibilities to

  4. An advanced approach for measuring acidity of hydroxyls in confined space: a FTIR study of low-temperature CO and (15)N2 adsorption on MOF samples from the MIL-53(Al) series.

    PubMed

    Mihaylov, M; Andonova, S; Chakarova, K; Vimont, A; Ivanova, E; Drenchev, N; Hadjiivanov, K

    2015-10-07

    Acidity of solids is decisive for their interaction with guest molecules. One of the most used methods for measuring the acidity of surface hydroxyl groups is the hydrogen bond method based on the spectral shift of the OH stretching modes induced by the adsorption of weak bases. However, many materials of practical interest (e.g. metal organic frameworks, zeolites, etc.) are porous and the OH groups are involved in H-bonding with framework basic sites. Here we show that MIL-53(Al) and NH2-MIL-53(Al) samples are characterized by one type of structural hydroxyl but three IR bands are detected at 100 K with these materials (at 3721, 3711 and 3683 cm(-1)). These bands are assigned to structural hydroxyls involved in H-bonding with different strengths. There is no correlation between the acidities of the hydroxyls, as measured by low-temperature CO or (15)N2 adsorption, and the main reason for this is the pre-existing H-bond. A method for the estimation of the intrinsic frequency of the OH groups (i.e. if not participating in H-bonds), based on the analysis of the spectral data obtained with two molecular probes, is proposed. According to this method, the OH stretching frequency of the structural hydroxyls of MIL-53(Al) samples is determined to be 3727 cm(-1). The formation of 1 : 1 adducts between the hydroxyls and strong bases leads to breaking of the pre-existing H-bonds. When the base is weak, bifurcated complexes are formed which slightly affects the spectral shift. The conclusions derived here considerably broaden the applicability of the H-bond method for assessing protonic acidity of materials and systems where the OH groups are preliminarily involved in H-bonding.

  5. Simplifying healthful choices: a qualitative study of a physical activity based nutrition label format

    PubMed Central

    2013-01-01

    Background This study used focus groups to pilot and evaluate a new nutrition label format and refine the label design. Physical activity equivalent labels present calorie information in terms of the amount of physical activity that would be required to expend the calories in a specified food item. Methods Three focus groups with a total of twenty participants discussed food choices and nutrition labeling. They provided information on comprehension, usability and acceptability of the label. A systematic coding process was used to apply descriptive codes to the data and to identify emerging themes and attitudes. Results Participants in all three groups were able to comprehend the label format. Discussion about label format focused on issues including gender of the depicted figure, physical fitness of the figure, preference for walking or running labels, and preference for information in miles or minutes. Feedback from earlier focus groups was used to refine the labels in an iterative process. Conclusions In contrast to calorie labels, participants shown physical activity labels asked and answered, “How does this label apply to me?” This shift toward personalized understanding may indicate that physical activity labels offer an advantage over currently available nutrition labels. PMID:23742678

  6. Simplifying healthful choices: a qualitative study of a physical activity based nutrition label format.

    PubMed

    Swartz, Jonas J; Dowray, Sunaina; Braxton, Danielle; Mihas, Paul; Viera, Anthony J

    2013-06-06

    This study used focus groups to pilot and evaluate a new nutrition label format and refine the label design. Physical activity equivalent labels present calorie information in terms of the amount of physical activity that would be required to expend the calories in a specified food item. Three focus groups with a total of twenty participants discussed food choices and nutrition labeling. They provided information on comprehension, usability and acceptability of the label. A systematic coding process was used to apply descriptive codes to the data and to identify emerging themes and attitudes. Participants in all three groups were able to comprehend the label format. Discussion about label format focused on issues including gender of the depicted figure, physical fitness of the figure, preference for walking or running labels, and preference for information in miles or minutes. Feedback from earlier focus groups was used to refine the labels in an iterative process. In contrast to calorie labels, participants shown physical activity labels asked and answered, "How does this label apply to me?" This shift toward personalized understanding may indicate that physical activity labels offer an advantage over currently available nutrition labels.

  7. Acid-base interactions and secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR and Ab initio model calculations.

    PubMed

    Dos, Alexandra; Schimming, Volkmar; Tosoni, Sergio; Limbach, Hans-Heinrich

    2008-12-11

    The interactions of the 15N-labeled amino groups of dry solid poly-L-lysine (PLL) with various halogen and oxygen acids HX and the relation to the secondary structure have been studied using solid-state 15N and 13C CPMAS NMR spectroscopy (CP = cross polarization and MAS = magic angle spinning). For comparison, 15N NMR spectra of an aqueous solution of PLL were measured as a function of pH. In order to understand the effects of protonation and hydration on the 15N chemical shifts of the amino groups, DFT and chemical shielding calculations were performed on isolated methylamine-acid complexes and on periodic halide clusters of the type (CH3NH3(+)X(-))n. The combined experimental and computational results reveal low-field shifts of the amino nitrogens upon interaction with the oxygen acids HX = HF, H2SO4, CH3COOH, (CH3)2POOH, H3PO4, HNO3, and internal carbamic acid formed by reaction of the amino groups with gaseous CO2. Evidence is obtained that only hydrogen-bonded species of the type (Lys-NH2***H-X)n are formed in the absence of water. 15N chemical shifts are maximum when H is located in the hydrogen bond center and then decrease again upon full protonation, as found for aqueous solution at low pH. By contrast, halogen acids interact in a different way. They form internal salts of the type (Lys-NH3(+)X(-))n via the interaction of many acid-base pairs. This salt formation is possible only in the beta-sheet conformation. By contrast, the formation of hydrogen-bonded complexes can occur both in beta-sheet domains as well as in alpha-helical domains. The 15N chemical shifts of the protonated ammonium groups increase when the size of the interacting halogen anions is increased from chloride to iodide and when the number of the interacting anions is increased. Thus, the observed high-field 15N shift of ammonium groups upon hydration is the consequence of replacing interacting halogen atoms by oxygen atoms.

  8. Isotopic labeling of proteins in Halobacterium salinarum.

    PubMed

    Cleveland, Thomas E; Kelman, Zvi

    2015-01-01

    It is often necessary to obtain isotopically labeled proteins containing (15)N, (13)C, or (2)H for nuclear magnetic resonance; and (2)H for small-angle neutron scattering or neutron diffraction studies. To achieve uniform isotopic labeling, protein expression is most commonly performed in Escherichia coli or yeast using labeled media. However, proteins from extreme halophiles sometimes require a cellular environment with high ionic strength and cannot be heterologously expressed in E. coli or yeast in functional form. We present here methods for the cultivation of Halobacterium salinarum in isotopically labeled rich media, using commercially available isotopically labeled hydrolysates. The methods described here are both technically simple and relatively inexpensive. 2015 Published by Elsevier Inc.

  9. Spatial variations in larch needle and soil δ(15)N at a forest-grassland boundary in northern Mongolia.

    PubMed

    Fujiyoshi, Lei; Sugimoto, Atsuko; Tsukuura, Akemi; Kitayama, Asami; Lopez Caceres, M Larry; Mijidsuren, Byambasuren; Saraadanbazar, Ariunaa; Tsujimura, Maki

    2017-03-01

    The spatial patterns of plant and soil δ(15)N and associated processes in the N cycle were investigated at a forest-grassland boundary in northern Mongolia. Needles of Larix sibirica Ledeb. and soils collected from two study areas were analysed to calculate the differences in δ(15)N between needle and soil (Δδ(15)N). Δδ(15)N showed a clear variation, ranging from -8 ‰ in the forest to -2 ‰ in the grassland boundary, and corresponded to the accumulation of organic layer. In the forest, the separation of available N produced in the soil with (15)N-depleted N uptake by larch and (15)N-enriched N immobilization by microorganisms was proposed to cause large Δδ(15)N, whereas in the grassland boundary, small Δδ(15)N was explained by the transport of the most available N into larch. The divergence of available N between larch and microorganisms in the soil, and the accumulation of diverged N in the organic layer control the variation in Δδ(15)N.

  10. Uptake of stormwater nitrogen in bioretention systems demonstrated from 15N tracer techniques

    NASA Astrophysics Data System (ADS)

    Houdeshel, D.; Hultine, K. R.; Pomeroy, C. A.

    2012-12-01

    Bioretention stormwater management systems are engineered ecosystems that capture urban stormwater in order to reduce the harmful effects of stormwater pollution on receiving waters. Bioretention systems have been shown to be effective at reducing the volume of runoff, and thereby reduce the nutrient loading to receiving waters from urban areas. However, little work has been done to evaluate the treatment processes that are responsible for reductions in effluent nitrogen (N). We hypothesize that the pulses of inorganic nitrogen associated with urban runoff events are captured in the plat tissues within these systems and not adsorbed to the soil media, thus creating a long-term, sustainable treatment approach to reducing the total nutrient loading to receiving waters. Nitrogen treatment performance was tested on two bioretention systems in Salt Lake City, UT: 1) an upland native community that does not require irrigation in semi-arid climates, and 2) a wetland community that requires 250 l of daily irrigation to offset the relatively high evaporative demand in the region. Each cell is sized to treat a 2.5 cm storm from a 140 m2 impervious surface: the area of the bioretention system is 10 m2. To test the N removal performance of each system, runoff events were simulated to represent an average precipitation regime using a synthetic stormwater blend starting in January, 2012. Effluent was collected from an underdrain and analyzed for total nitrogen (TN); mass removal was calculated for each month by subtracting the TN mass added to the garden minus the TN mass that flowed out of the garden. To test the hypothesis that plants assimilate stormwater N, 4 g of 100 atom% 15N NH4NO3 tracer was used as the N source in the synthetic stormwater during the first 2,000 l synthetic storm event in May. This isotopic label was calculated to enrich the total N pool of each garden to 100‰ 15N/14Nair. New growth was harvested from each plant in both cells and analyzed for 15N

  11. Nitrogen source tracking with delta(15)N content of coastal wetland plants in Hawaii.

    PubMed

    Bruland, Gregory L; MacKenzie, Richard A

    2010-01-01

    Inter- and intra-site comparisons of the nitrogen (N) stable isotope composition of wetland plant species have been used to identify sources of N in coastal areas. In this study, we compared delta(15)N values from different herbaceous wetland plants across 34 different coastal wetlands from the five main Hawaiian Islands and investigated relationships of delta(15)N with land use, human population density, and surface water quality parameters (i.e., nitrate, ammonium, and total dissolved N). The highest delta(15)N values were observed in plants from wetlands on the islands of Oahu (8.7-14.6 per thousand) and Maui (8.9-9.2 per thousand), whereas plants from wetlands on the islands of Kauai, Hawaii, and Molokai had delta(15)N values usually <4 per thousand. The enrichment in delta(15)N values in plant tissues from wetlands on Oahu and Maui was most likely a result of the more developed and densely populated watersheds on these two islands. Urban development within a 1000-m radius and population density were positively correlated to average delta(15)N vegetation values from each wetland site (r = 0.56 and 0.51, respectively; p < 0.001). This suggested that site mean delta(15)N values from mixed stands of wetland plants have potential as indices of N sources in coastal lowland wetlands in Hawaii and that certain sites on Oahu and Maui have experienced significant anthropogenic N loading. This information can be used to monitor future changes in N inputs to coastal wetlands throughout Hawaii and the Pacific.

  12. The instrumental role of product information: a study of warning labels for non-prescription drugs.

    PubMed

    Discenza, R; Ferguson, J M

    1992-01-01

    The study extends work in informative labeling, fear appeals, and negative information effects. Respondents were given two labels from two packages, one of which contained the experimental treatment. Warning strength was manipulated at three levels: weak, medium, and strong. The data show that, unlike labels on prescription medications, non-prescription warning labels tend to discourage use of the product. Results have implications for information theorists, marketers, and public policy makers.

  13. Rivermouth Alteration of Agricultural Impacts on Consumer Tissue δ15N

    PubMed Central

    Larson, James H.; Richardson, William B.; Vallazza, Jon M.; Nelson, John C.

    2013-01-01

    Terrestrial agricultural activities strongly influence riverine nitrogen (N) dynamics, which is reflected in the δ15N of riverine consumer tissues. However, processes within aquatic ecosystems also influence consumer tissue δ15N. As aquatic processes become more important terrestrial inputs may become a weaker predictor of consumer tissue δ15N. In a previous study, this terrestrial-consumer tissue δ15N connection was very strong at river sites, but was disrupted by processes occurring in rivermouths (the ‘rivermouth effect’). This suggested that watershed indicators of N loading might be accurate in riverine settings, but could be inaccurate when considering N loading to the nearshore of large lakes and oceans. In this study, the rivermouth effect was examined on twenty-five sites spread across the Laurentian Great Lakes. Relationships between agriculture and consumer tissue δ15N occurred in both upstream rivers and at the outlets where rivermouths connect to the nearshore zone, but agriculture explained less variation and had a weaker effect at the outlet. These results suggest that rivermouths may sometimes be significant sources or sinks of N, which would cause N loading estimates to the nearshore zone that are typically made at discharge gages further upstream to be inaccurate. Identifying definitively the controls over the rivermouth effect on N loading (and other nutrients) will require integration of biogeochemical and hydrologic models. PMID:23935980

  14. Rivermouth alteration of agricultural impacts on consumer tissue δ(15)N.

    PubMed

    Larson, James H; Richardson, William B; Vallazza, Jon M; Nelson, John C

    2013-01-01

    Terrestrial agricultural activities strongly influence riverine nitrogen (N) dynamics, which is reflected in the δ(15)N of riverine consumer tissues. However, processes within aquatic ecosystems also influence consumer tissue δ(15)N. As aquatic processes become more important terrestrial inputs may become a weaker predictor of consumer tissue δ(15)N. In a previous study, this terrestrial-consumer tissue δ(15)N connection was very strong at river sites, but was disrupted by processes occurring in rivermouths (the 'rivermouth effect'). This suggested that watershed indicators of N loading might be accurate in riverine settings, but could be inaccurate when considering N loading to the nearshore of large lakes and oceans. In this study, the rivermouth effect was examined on twenty-five sites spread across the Laurentian Great Lakes. Relationships between agriculture and consumer tissue δ(15)N occurred in both upstream rivers and at the outlets where rivermouths connect to the nearshore zone, but agriculture explained less variation and had a weaker effect at the outlet. These results suggest that rivermouths may sometimes be significant sources or sinks of N, which would cause N loading estimates to the nearshore zone that are typically made at discharge gages further upstream to be inaccurate. Identifying definitively the controls over the rivermouth effect on N loading (and other nutrients) will require integration of biogeochemical and hydrologic models.

  15. Rivermouth alteration of agricultural impacts on consumer tissue δ15N

    USGS Publications Warehouse

    Larson, James H.; Richardson, William B.; Vallazza, Jonathan M.; Nelson, J. C.

    2013-01-01

    Terrestrial agricultural activities strongly influence riverine nitrogen (N) dynamics, which is reflected in the δ15N of riverine consumer tissues. However, processes within aquatic ecosystems also influence consumer tissue δ15N. As aquatic processes become more important terrestrial inputs may become a weaker predictor of consumer tissue δ15N. In a previous study, this terrestrial-consumer tissue δ15N connection was very strong at river sites, but was disrupted by processes occurring in rivermouths (the ‘rivermouth effect’). This suggested that watershed indicators of N loading might be accurate in riverine settings, but could be inaccurate when considering N loading to the nearshore of large lakes and oceans. In this study, the rivermouth effect was examined on twenty-five sites spread across the Laurentian Great Lakes. Relationships between agriculture and consumer tissue δ15N occurred in both upstream rivers and at the outlets where rivermouths connect to the nearshore zone, but agriculture explained less variation and had a weaker effect at the outlet. These results suggest that rivermouths may sometimes be significant sources or sinks of N, which would cause N loading estimates to the nearshore zone that are typically made at discharge gages further upstream to be inaccurate. Identifying definitively the controls over the rivermouth effect on N loading (and other nutrients) will require integration of biogeochemical and hydrologic models.

  16. Delta15N values of tropical savanna and monsoon forest species reflect root specialisations and soil nitrogen status.

    PubMed

    Schmidt, S; Stewart, G R

    2003-03-01

    A large number of herbaceous and woody plants from tropical woodland, savanna, and monsoon forest were analysed to determine the impact of environmental factors (nutrient and water availability, fire) and biological factors (microbial associations, systematics) on plant delta(15)N values. Foliar delta(15)N values of herbaceous and woody species were not related to growth form or phenology, but a strong relationship existed between mycorrhizal status and plant delta(15)N. In woodland and savanna, woody species with ectomycorrhizal (ECM) associations and putative N(2)-fixing species with ECM/arbuscular (AM) associations had lowest foliar delta(15)N values (1.0-0.6 per thousand ), AM species had mostly intermediate delta(15)N values (average +0.6 per thousand ), while non-mycorrhizal Proteaceae had highest delta(15)N values (+2.9 to +4.1 per thousand ). Similar differences in foliar delta(15)N were observed between AM (average 0.1 and 0.2 per thousand ) and non-mycorrhizal (average +0.8 and +0.3 per thousand ) herbaceous species in woodland and savanna. Leguminous savanna species had significantly higher leaf N contents (1.8-2.5% N) than non-fixing species (0.9-1.2% N) indicating substantial N acquisition via N(2) fixation. Monsoon forest species had similar leaf N contents (average 2.4% N) and positive delta(15)N values (+0.9 to +2.4 per thousand ). Soil nitrification and plant NO(3)(-) use was substantially higher in monsoon forest than in woodland or savanna. In the studied communities, higher soil N content and nitrification rates were associated with more positive soil delta(15)N and plant delta(15)N. In support of this notion, Ficus, a high NO(3)(-) using taxa associated with NO(3)(-) rich sites in the savanna, had the highest delta(15)N values of all AM species in the savanna. delta(15)N of xylem sap was examined as a tool for studying plant delta(15)N relations. delta(15)N of xylem sap varied seasonally and between differently aged Acacia and other savanna

  17. Probing platinum azido complexes by 14N and 15N NMR spectroscopy.

    PubMed

    Farrer, Nicola J; Gierth, Peter; Sadler, Peter J

    2011-10-17

    Metal azido complexes are of general interest due to their high energetic properties, and platinum azido complexes in particular because of their potential as photoactivatable anticancer prodrugs. However, azido ligands are difficult to probe by NMR spectroscopy due to the quadrupolar nature of (14)N and the lack of scalar (1)H coupling to enhance the sensitivity of the less abundant (15)N by using polarisation transfer. In this work, we report (14)N and (15)N NMR spectroscopic studies of cis,trans,cis-[Pt(N(3))(2)(OH)(2)(NH(3))] (1) and trans,trans,trans-[Pt(N(3))(2)(OH)(2)(X)(Y)], where X=Y=NH(3) (2); X=NH(3), Y=py (3) (py=pyridine); X=Y=py (4); and selected Pt(II) precursors. These studies provide the first (15)N NMR data for azido groups in coordination complexes. We discuss one- and three-bond J((15)N,(195)Pt) couplings for azido and am(m)ine ligands. The (14)N(α) (coordinated azido nitrogen) signal in the Pt(IV) azido complexes is extremely broad (W(1/2)≈2124 Hz for 4) in comparison to other metal azido complexes, attributable to a highly asymmetrical electric field gradient at the (14)N(α) atom. Through the use of anti-ringing pulse sequences, the (14)N NMR spectra, which show resolution of the broad (14)N(α) peak, were obtained rapidly (e.g., 1.5 h for 10 mM 4). The linewidths of the (14)N(α) signals correlate with the viscosity of the solvent. For (15) N-enriched samples, it is possible to detect azido (15)N resonances directly, which will allow photoreactions to be followed by 1D (15)N NMR spectroscopy. The T(1) relaxation times for 3 and 4 were in the range 5.7-120 s for (15)N, and 0.9-11.3 ms for (14)N. Analysis of the (1)J((15)N,(195)Pt) coupling constants suggests that an azido ligand has a moderately strong trans influence in octahedral Pt(IV) complexes, within the series 2-pic

  18. Plant and Soil Natural Abundance delta-15N: Indicators of Nitrogen Cycling in the Catskill Mountains, New York, USA

    NASA Astrophysics Data System (ADS)

    Templer, P. H.; Lovett, G. M.; Weathers, K.; Arthur, M. A.

    2002-12-01

    We examined the potential use of natural abundance 15N of plants and soils as an indicator of forest nitrogen (N) cycling rates within the Catskill Mountains, NY. These watersheds receive among the highest rates of N deposition in the northeastern United States and are beginning to show signs of N saturation. Many studies have shown a link between increased N cycling rates and 15N enrichment of soil and plant pools. Faster rates of N cycling processes, especially nitrification, lead to fractionation of 14/15N, creating N products that are relatively depleted in 15N. This can lead to enrichment of soil pools, as lighter 14N is lost from the system via leaching or denitrification. Plant N pools can become increasingly enriched as they take up 15N-enriched soil N. Despite similar amounts of N deposition across the Catskill Mountains, forests dominated by different tree species appear to vary in the amount of N retained or lost to nearby streams. To determine if plant and soil 15N could be used as indicators of N cycling rates, we collected foliage, wood, litterfall, organic and mineral soil, and fine roots from single species stands of American beech (Fagus grandifolia), eastern hemlock (Tsuga canadensis), red oak (Quercus rubra), and sugar maple (Acer saccharum). Fine roots and soil 15N were highest within sugar maple stands (p<0.05). Sugar maple soils also had the highest rates of net nitrification and N leaching. Therefore, soil 15N appears to correlate with forest N retention and loss. However, 15N enrichment was highest within foliage, litterfall and wood of beech trees (p<0.05). The decoupling between foliage 15N and N cycling, as well as between 15N of foliage and fine roots, illustrates that it may not be possible to use a single plant pool as an indicator of N cycling rates.

  19. Can δ(15)N in lettuce tissues reveal the use of synthetic nitrogen fertiliser in organic production?

    PubMed

    Sturm, Martina; Kacjan-Maršić, Nina; Lojen, Sonja

    2011-01-30

    The nitrogen isotopic fingerprint (δ(15)N) is reported to be a promising indicator for differentiating between organically and conventionally grown vegetables. However, the effect on plant δ(15)N of split nitrogen fertilisation, which could enable farmers to cover up the use of synthetic fertiliser, is not well studied. In this study the use of δ(15)N in lettuce as a potential marker for identifying the use of synthetic nitrogen fertiliser was tested on pot-grown lettuce (Lactuca sativa L.) treated with synthetic and organic nitrogen fertilisers (single or split application). The effect of combined usage of synthetic and organic fertilisers on δ(15)N was also investigated. The δ(15)N values of whole plants treated with different fertilisers differed significantly when the fertiliser was applied in a single treatment. However, additional fertilisation (with isotopically the same or different fertiliser) did not cause a significant alteration of plant δ(15)N. The findings of the study suggest that the δ(15)N value of lettuce tissues could be used as a rough marker to reveal the history of nitrogen fertilisation, but only in the case of single fertiliser application. However, if the difference in δ(15)N between the applied synthetic and organic nitrogen fertilisers was > 9.1 ‰, the detection of split and combined usage of the fertilisers would have greater discriminatory power. 2010 Society of Chemical Industry.

  20. A new method to track seed dispersal and recruitment using 15N isotope enrichment.

    PubMed

    Carlo, Tomás A; Tewksbury, Joshua J; Martínez Del Río, Carlos

    2009-12-01

    Seed dispersal has a powerful influence on population dynamics, genetic structuring, evolutionary rates, and community ecology. Yet, patterns of seed dispersal are difficult to measure due to methodological shortcomings in tracking dispersed seeds from sources of interest. Here we introduce a new method to track seed dispersal: stable isotope enrichment. It consists of leaf-feeding plants with sprays of 15N-urea during the flowering stage such that seeds developed after applications are isotopically enriched. We conducted a greenhouse experiment with Solanum americanum and two field experiments with wild Capsicum annuum in southern Arizona, USA, to field-validate the method. First, we show that plants sprayed with 15N-urea reliably produce isotopically enriched progeny, and that delta 15N (i.e., the isotopic ratio) of seeds and seedlings is a linear function of the 15N-urea concentration sprayed on mothers. We demonstrate that three urea dosages can be used to distinctly enrich plants and unambiguously differentiate their offspring after seeds are dispersed by birds. We found that, with high urea dosages, the resulting delta 15N values in seedlings are 10(3) - 10(4) times higher than the delta 15N values of normal plants. This feature allows tracking not only where seeds arrive, but in locations where seeds germinate and recruit, because delta 15N enrichment is detectable in seedlings that have increased in mass by at least two orders of magnitude before fading to normal delta 15N values. Last, we tested a mixing model to analyze seed samples in bulk. We used the delta 15N values of batches (i.e., combined seedlings or seeds captured in seed traps) to estimate the number of enriched seeds coming from isotopically enriched plants in the field. We confirm that isotope enrichment, combined with batch-sampling, is a cheap, reliable, and user-friendly method for bulk-processing seeds and is thus excellent for the detection of rare dispersal events. This method could

  1. Nitrogen and 15N in the Mer Bleue peatland

    NASA Astrophysics Data System (ADS)

    Moore, Tim

    2017-04-01

    Although much of our attention in peatlands has focussed on carbon, as CO2, CH4 and DOC processing and fluxes, N plays an important role in the functioning of these ecosystems. Here, I present information on the distribution of N and 15N in plant and peat tissues and relate them to the cycling of N. N concentration in foliar tissues, ranged from 0.67 to 1.3% in evergreen shrubs and trees and mosses with little seasonal variation, and with a strong seasonal variation from 0.5 to 3.5% in the deciduous forbs, shrubs and trees, with a strong overall relationship to [chlorophyll]. Although the proportion of shrubs and mosses varied with microtopography the spatial foliar mass of N varied little with water table position, resulting in minor spatial variations in photosynthetic potential. Decomposition of plant tissues through litter to peat resulted in a decrease in the C:N ratio from about 50:1 to about 30:1 at the base of the profile, representing peat about 8000 yr old. This marginally larger loss of N through decomposition (mainly as TDN, 0.4 g N m-2 yr-1) compared to C produced a long-term N accumulation rate of 0.9 g N m-2 yr-1, being smaller in the bog phase, 0.6 N m-2 yr-1, and over past 150 yr, 0.8 g N m-2 yr-1. Although N is 'hard won' through N2 fixation, northern peatlands are significant global sinks of N and have limited N availability. del15N in foliar tissues ranged from -4 to -9 ‰ in evergreen and deciduous shrubs and trees, from -4 to -5 ‰ in mosses and from -1 to +1 ‰ in sedges and forbs. This appears to be a function of the mycorhizzal infection of the shrubs and trees, compared to sedges and forbs and the values for mosses may partially reflect the signature of atmospheric N deposition. There was no strong correlation between foliar [N] and del15N. In peat profiles from bog and fen sections of Mer Bleue, del15N values in peat fell from -5 to -2 ‰ in the top 10 cm to values of -1 to +1 ‰ at a depth of 40 cm and remained close to 0 ‰ below

  2. Heating Isotopically Labeled Bernal Stacked Graphene: A Raman Spectroscopy Study.

    PubMed

    Ek-Weis, Johan; Costa, Sara; Frank, Otakar; Kalbac, Martin

    2014-02-06

    One of the greatest issues of nanoelectronics today is how to control the heating of the components. Graphene is a promising material in this area, and it is essential to study its thermal properties. Here, the effect of heating a bilayer structure was investigated using in situ Raman spectroscopy. In order to observe the effects on each individual layer, an isotopically labeled bilayer graphene was synthesized where the two layers were composed of different carbon isotopes. Therefore, the frequency of the phonons in the Raman spectra was shifted in relation to each other. This technique was used to investigate the influence of different stacking order. It was found that in bilayer graphene grown by chemical vapor deposition (CVD), the two layers behave very similarly for both Bernal stacking and randomly oriented structures, while for transferred samples, the layers act more independently. This highlights a significant dependence on the sample preparation procedure.

  3. How do design features influence consumer attention when looking for nutritional information on food labels? Results from an eye-tracking study on pan bread labels.

    PubMed

    Antúnez, Lucía; Vidal, Leticia; Sapolinski, Alejandra; Giménez, Ana; Maiche, Alejandro; Ares, Gastón

    2013-08-01

    The aim of this work was to evaluate consumer visual processing of food labels when evaluating the salt content of pan bread labels and to study the influence of label design and nutritional labelling format on consumer attention. A total of 16 pan bread labels, designed according to a full factorial design, were presented to 52 participants, who were asked to decide whether the sodium content of each label was medium or low, while their eye movements were recorded using an eye tracker. Results showed that most participants looked at nutrition labels and the traffic light system to conclude on the salt content of the labels. However, the average percentage of participants who looked at the actual sodium content was much lower. Nutrition information format affected participants' processing of nutrition information. Among other effects, the inclusion of the traffic light system increased participants' attention towards some kind of nutrition information and facilitated its processing, but not its understanding.

  4. A study on the consumer's perception of front-of-pack nutrition labeling

    PubMed Central

    Kim, Woo Kyoung

    2009-01-01

    The goal of this research is to investigate the present situation for front of pack labeling in Korea and the perception of consumers for the new system of labeling, front of pack labeling, based on the consumer survey. We investigated the number of processed foods with front of pack labeling in one retailer in Youngin-si. And we also surveyed 1,019 participants nationwide whose ages were from 20 to 49; the knowledge of nutrition labeling, the knowledge of 'front of pack labeling', and the opinion about the labeling system. The data were analyzed using SAS statistics program. The results were as follows: 13.4% of processed foods had front of pack labeling, and 16.8% of the consumers always checked the nutrition labeling, while 32.7% of the consumers seldom checked it. In addition, 44.3% of the consumers think that 'front of pack labeling' is necessary, and 58.3% of the consumers think it is important to show the percentage of daily value as a way of 'front of pack labeling'. However, 32% of the consumer think the possibility of 'front of pack labeling' is slim. Meanwhile, 58.3% of the consumers think that it is important to have the color difference according to contents. The number of favorite nutrients in the front of pack was four or five. It seems that the recognition of current nutrition labeling has the influence on the willingness of using the future 'front of pack labeling'. Along with our study, the policy for 'front of pack labeling' has to be updated and improved constantly since 'front of pack labeling' helps consumer understand nutrition facts. PMID:20098583

  5. Promoting public health through nutrition labeling - a study in Brazil.

    PubMed

    Souza, Sônia Maria Fernandes da Costa; Lima, Kenio Costa; Alves, Maria do Socorro Costa Feitosa

    2016-01-01

    Food and nutrition education allows individuals to build knowledge and values, reframe their food practices, and develop strategies for a healthy diet. Food choices within the diet represent a determinant of individual health status. Regardless of the food quality, the consumption of calorie-dense foods does not promote better health conditions for the population and can worsen emerging health problems. The present study aimed to describe and analyze the effectiveness of educational activities related to nutrition information for enabling healthy food choices, as a tool to promote public health. To describe and analyze the effectiveness of an educational intervention regarding nutrition labeling as a tool to promote healthy food choices, 702 individuals were enrolled in the present quasi-experimental study. The Wilcoxon and McNemar tests were used to compare the pre- and post-intervention data, and a p value <0.05 was considered statistically significant. Of the 702 participants (mean age, 26.6 years), 17.4 % were male, and 82.6 % were female. The education level was high school for 53.2 % of the participants. The mean income was R$ 1969.54 (about 500 USD). In the pre-test, 55.8 % of the respondents reported consulting the nutrition information provided on packaged foods. At the post-test, 72.0 % of respondents reported consulting this information (p < 0.001; Table 1). However, the change in the response regarding the purchase of packaged products was borderline significant. The results indicate that the intervention was feasible and acceptable and improved knowledge regarding the role of nutrition labeling in promoting healthy eating. These results support the importance of an educational intervention to reinforce healthy food choices.

  6. 14N15N detectability in Pluto’s atmosphere

    NASA Astrophysics Data System (ADS)

    Jessup, Kandis Lea; Gladstone, G. R.; Heays, A. N.; Gibson, S. T.; Lewis, B. R.; Stark, G.

    2013-11-01

    Based on the vapor pressure behavior of Pluto’s surface ices, Pluto’s atmosphere is expected to be predominantly composed of N2 gas. Measurement of the N2 isotopologue 15N/14N ratio within Pluto’s atmosphere would provide important clues to the evolution of Pluto’s atmosphere from the time of formation to its present state. The most straightforward way of determining the N2 isotopologue 15N/14N ratio in Pluto’s atmosphere is via spectroscopic observation of the 14N15N gas species. Recent calculations of the 80-100 nm absorption behavior of the 14N2 and 14N15N isotopologues by Heays et al. (Heays, A.N. et al. [2011]. J. Chem. Phys. 135, 244301), Lewis et al. (Lewis, B.R., Heays, A.N., Gibson, S.T., Lefebvre-Brion, H., Lefebvre, R. [2008]. J. Chem. Phys. 129, 164306); Lewis et al. (Lewis, B.R., Gibson, S.T., Zhang, W., Lefebvre-Brion, H., Robbe, J.-M. [2005]. J. Chem. Phys. 122, 144302), and Haverd et al. (Haverd, V.E., Lewis, B.R., Gibson, S.T., Stark, G. [2005]. J. Chem. Phys. 123, 214304) show that the peak magnitudes of the 14N2 and 14N15N absorption bandhead cross-sections are similar, but the locations of the bandhead peaks are offset in wavelength by ∼0.05-0.1 nm. These offsets make the segregation of the 14N2 and 14N15N absorption signatures possible. We use the most recent N2 isotopologue absorption cross-section calculations and the atmospheric density profiles resulting from photochemical models developed by Krasnopolsky and Cruickshank (Krasnopolsky, V.A., Cruickshank, D.P. [1999]. J. Geophys. Res. 104, 21979-21996) to predict the level of solar light that will be transmitted through Pluto’s atmosphere as a function of altitude during a Pluto solar occultation. We characterize the detectability of the isotopic absorption signature per altitude assuming 14N15N concentrations ranging from 0.1% to 2% of the 14N2 density and instrumental spectral resolutions ranging from 0.01 to 0.3 nm. Our simulations indicate that optical depth of unity is

  7. Intracellular Isotope Localization in Ammonia sp. (Foraminifera) of Oxygen-Depleted Environments: Results of Nitrate and Sulfate Labeling Experiments.

    PubMed

    Nomaki, Hidetaka; Bernhard, Joan M; Ishida, Akizumi; Tsuchiya, Masashi; Uematsu, Katsuyuki; Tame, Akihiro; Kitahashi, Tomo; Takahata, Naoto; Sano, Yuji; Toyofuku, Takashi

    2016-01-01

    Some benthic foraminiferal species are reportedly capable of nitrate storage and denitrification, however, little is known about nitrate incorporation and subsequent utilization of nitrate within their cell. In this study, we investigated where and how much (15)N or (34)S were assimilated into foraminiferal cells or possible endobionts after incubation with isotopically labeled nitrate and sulfate in dysoxic or anoxic conditions. After 2 weeks of incubation, foraminiferal specimens were fixed and prepared for Transmission Electron Microscopy (TEM) and correlative nanometer-scale secondary ion mass spectrometry (NanoSIMS) analyses. TEM observations revealed that there were characteristic ultrastructural features typically near the cell periphery in the youngest two or three chambers of the foraminifera exposed to anoxic conditions. These structures, which are electron dense and ~200-500 nm in diameter and co-occurred with possible endobionts, were labeled with (15)N originated from (15)N-labeled nitrate under anoxia and were labeled with both (15)N and (34)S under dysoxia. The labeling with (15)N was more apparent in specimens from the dysoxic incubation, suggesting higher foraminiferal activity or increased availability of the label during exposure to oxygen depletion than to anoxia. Our results suggest that the electron dense bodies in Ammonia sp. play a significant role in nitrate incorporation and/or subsequent nitrogen assimilation during exposure to dysoxic to anoxic conditions.

  8. Intracellular Isotope Localization in Ammonia sp. (Foraminifera) of Oxygen-Depleted Environments: Results of Nitrate and Sulfate Labeling Experiments

    PubMed Central

    Nomaki, Hidetaka; Bernhard, Joan M.; Ishida, Akizumi; Tsuchiya, Masashi; Uematsu, Katsuyuki; Tame, Akihiro; Kitahashi, Tomo; Takahata, Naoto; Sano, Yuji; Toyofuku, Takashi

    2016-01-01

    Some benthic foraminiferal species are reportedly capable of nitrate storage and denitrification, however, little is known about nitrate incorporation and subsequent utilization of nitrate within their cell. In this study, we investigated where and how much 15N or 34S were assimilated into foraminiferal cells or possible endobionts after incubation with isotopically labeled nitrate and sulfate in dysoxic or anoxic conditions. After 2 weeks of incubation, foraminiferal specimens were fixed and prepared for Transmission Electron Microscopy (TEM) and correlative nanometer-scale secondary ion mass spectrometry (NanoSIMS) analyses. TEM observations revealed that there were characteristic ultrastructural features typically near the cell periphery in the youngest two or three chambers of the foraminifera exposed to anoxic conditions. These structures, which are electron dense and ~200–500 nm in diameter and co-occurred with possible endobionts, were labeled with 15N originated from 15N-labeled nitrate under anoxia and were labeled with both 15N and 34S under dysoxia. The labeling with 15N was more apparent in specimens from the dysoxic incubation, suggesting higher foraminiferal activity or increased availability of the label during exposure to oxygen depletion than to anoxia. Our results suggest that the electron dense bodies in Ammonia sp. play a significant role in nitrate incorporation and/or subsequent nitrogen assimilation during exposure to dysoxic to anoxic conditions. PMID:26925038

  9. A novel method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies

    NASA Astrophysics Data System (ADS)

    Wu, Dianming; Kampf, Christopher; Pöschl, Ulrich; Oswald, Robert; Cui, Junfang; Ermel, Michael; Hu, Chunsheng; Trebs, Ivonne; Sörgel, Matthias

    2014-05-01

    We developed a new method (gas-phase stripping-derivatization coupled to LC-MS) to measure the 15N atom percent excess (APE) of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye by the well-known Griess reaction in the Long Path Absorption Photometer (LOPAP). The reaction solutions containing the dye are collected at the outflow of the LOPAP, purified by solid-phase extraction and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The unlabeled azo dye (C18H19O2N5S) with a monoisotopic molecular mass of 369.41 g mol-1 can be detected as its protonated molecular ion ([M+H+], M) by HPLC-MS at a retention time of 2.8 min. Due to the natural isotope distribution M + 0, M + 1, M + 2, and M + 3 ions were considered for the calculation of the 15N APE. The optimal working range was found to be between 20 and 50% for the 15N/14N ratio. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method has been applied for the measurement of HO15NO emissions from soil in a dynamic chamber with and without spiking 15N labeled urea. Our results confirm biogenic HONO emissions from soil as HO15NO was measured after addition of 15N urea.

  10. Macroalgae δ15N values in well-mixed estuaries: Indicator of anthropogenic nitrogen input or macroalgae metabolism?

    NASA Astrophysics Data System (ADS)

    Raimonet, Mélanie; Guillou, Gaël; Mornet, Françoise; Richard, Pierre

    2013-03-01

    Although nitrogen stable isotope ratio (δ15N) in macroalgae is widely used as a bioindicator of anthropogenic nitrogen inputs to the coastal zone, recent studies suggest the possible role of macroalgae metabolism in δ15N variability. Simultaneous determinations of δ15N of dissolved inorganic nitrogen (DIN) along the land-sea continuum, inter-species variability of δ15N and its sensitivity to environmental factors are necessary to confirm the efficiency of macroalgae δ15N in monitoring nitrogen origin in mixed-use watersheds. In this study, δ15N of annual and perennial macroalgae (Ulva sp., Enteromorpha sp., Fucus vesiculosus and Fucus serratus) are compared to δ15N-DIN along the Charente Estuary, after characterizing δ15N of the three main DIN sources (i.e. cultivated area, pasture, sewage treatment plant outlet). During late winter and spring, when human activities produce high DIN inputs, DIN sources exhibit distinct δ15N signals in nitrate (NO) and ammonium (NH): cultivated area (+6.5 ± 0.6‰ and +9.0 ± 11.0‰), pasture (+9.2 ± 1.8‰ and +12.4‰) and sewage treatment plant discharge (+16.9 ± 8.7‰ and +25.4 ± 5.9‰). While sources show distinct δN- in this multiple source catchment, the overall mixture of NO sources - generally >95% DIN - leads to low variations of δN-NO at the mouth of the estuary (+7.7 to +8.4‰). Even if estuarine δN-NO values are not significantly different from pristine continental and oceanic site (+7.3‰ and +7.4‰), macroalgae δ15N values are generally higher at the mouth of the estuary. This highlights high anthropogenic DIN inputs in the estuary, and enhanced contribution of 15N-depleted NH in oceanic waters. Although seasonal variations in δN-NO are low, the same temporal trends in macroalgae δ15N values at estuarine and oceanic sites, and inter-species differences in δ15N values, suggest that macroalgae δ15N values might be modified by the metabolic response of macroalgae to environmental parameters (e

  11. 3D NMR Experiments for Measuring 15N Relaxation Data of Large Proteins: Application to the 44 kDa Ectodomain of SIV gp41

    NASA Astrophysics Data System (ADS)

    Caffrey, Michael; Kaufman, Joshua; Stahl, Stephen J.; Wingfield, Paul T.; Gronenborn, Angela M.; Clore, G. Marius

    1998-12-01

    A suite of 3D NMR experiments for measuring15N-{1H} NOE,15NT1, and15NT1ρvalues in large proteins, uniformly labeled with15N and13C, is presented. These experiments are designed for proteins that exhibit extensive spectral overlap in the 2D1H-15N HSQC spectrum. The pulse sequences are readily applicable to perdeuterated samples, which increases the spectral resolution and signal-to-noise ratio, thereby permitting the characterization of protein dynamics to be extended to larger protein systems. Application of the pulse sequences is demonstrated on a perdeuterated13C/15N-labeled sample of the 44 kDa ectodomain of SIV gp41.

  12. Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register.

    PubMed

    Frederick, Kendra K; Michaelis, Vladimir K; Caporini, Marc A; Andreas, Loren B; Debelouchina, Galia T; Griffin, Robert G; Lindquist, Susan

    2017-04-04

    The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of (13)C- and (15)N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems.

  13. Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

    PubMed Central

    Frederick, Kendra K.; Michaelis, Vladimir K.; Caporini, Marc A.; Debelouchina, Galia T.; Griffin, Robert G.; Lindquist, Susan

    2017-01-01

    The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13C- and 15N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. PMID:28330994

  14. An exploratory study of drinkers views of health information and warning labels on alcohol containers.

    PubMed

    Thomson, Lisa M; Vandenberg, Brian; Fitzgerald, John L

    2012-03-01

    To identify general and specific features of health information warning labels on alcohol beverage containers that could potentially inform the development and implementation of a new labelling regime in Australia. Mixed methods, including a cross-sectional population survey and a qualitative study of knowledge, attitudes and behaviours regarding alcohol beverage labelling. The population survey used computer-assisted telephone interviews of 1500 persons in Victoria, Australia to gauge the level of support for health information and warning labels. The qualitative study used six focus groups to test the suitability of 12 prototype labels that were placed in situ on a variety of alcohol beverage containers. The telephone survey found 80% to 90% support for a range of information that could potentially be mandated by government authorities for inclusion on labels (nutritional information, alcohol content, health warning, images). Focus group testing of the prototype label designs found that labels should be integrated with other alcohol-related health messages, such as government social advertising campaigns, and specific labels should be matched appropriately to specific consumer groups and beverage types. There are high levels of public support for health information and warning labels on alcohol beverages. This study contributes much needed empirical guidance for developing alcohol beverage labelling strategies in an Australian context. © 2011 Australasian Professional Society on Alcohol and other Drugs.

  15. Derivatives of pyrazinecarboxylic acid: 1H, 13C and 15N NMR spectroscopic investigations.

    PubMed

    Holzer, Wolfgang; Eller, Gernot A; Datterl, Barbara; Habicht, Daniela

    2009-07-01

    NMR spectroscopic studies are undertaken with derivatives of 2-pyrazinecarboxylic acid. Complete and unambiguous assignment of chemical shifts ((1)H, (13)C, (15)N) and coupling constants ((1)H,(1)H; (13)C,(1)H; (15)N,(1)H) is achieved by combined application of various 1D and 2D NMR spectroscopic techniques. Unequivocal mapping of (13)C,(1)H spin coupling constants is accomplished by 2D (delta,J) long-range INEPT spectra with selective excitation. Phenomena such as the tautomerism of 3-hydroxy-2-pyrazinecarboxylic acid are discussed.

  16. Enrichment of natural (15)N abundance during soil N losses under 20years of continuous cereal cropping.

    PubMed

    Jones, Andrew R; Dalal, Ram C

    2017-01-01

    It is generally accepted that the enrichment of natural (15)N abundance in soil over time is reflective of historic N cycling and loss, but this process in cropping soils is not yet clear. In this study, we identified an enrichment gradient of natural (15)N abundance during 20-year chronosequence of cereal cropping on Alfisols in southwest Queensland, Australia, that have no history of fertilisation. We demonstrate that the increase in soil (15)N abundance is explained by isotopic fractionation of (15)N during organic N mineralisation and nitrification, which lead to isotopically heavier ammonium retained in the soil and isotopically lighter soil nitrate taken up and removed by seasonal crops during harvest. Here we present a framework for natural (15)N isotopic fractionation co-occurring with N losses during long-term cultivation.

  17. Stepwise enrichment of 15N along food chains: Further evidence and the relation between δ 15N and animal age

    NASA Astrophysics Data System (ADS)

    Minagawa, Masao; Wada, Eitaro

    1984-05-01

    The isotopic composition of nitrogen was measured in marine and fresh-water animals from the East China Sea, The Bering Sea, Lake Ashinoko and Usujiri intertidal zone. Primary producers, showed average δ15Nversus atmospheric nitrogen of +5.0%. (+3.4 to +7.5) in the Bering Sea and Lake Ashinoko, and +6.8%. (+6.0 to +7.6) in Usujiri intertidal zone. Blue green algae from the East China Sea show an average -0.55%. (-0.8 to +1.2). All consumers, Zooplankton, fish and bird exhibited Stepwise enrichment of 15N with increasing trophic level. The 15N enrichment at a single feeding process ranged from +1.3 to +5.3 averaging +3.4 ± 1.1%.. This isotopic fractionation seems to be independent of habitat. The effect of age in animals was obtained by analyzing two marine mussels. The soft tissue nitrogen showed +2.0%. enrichment relative to that of primary producers, and the magnitude was almost constant with shell ages ranging from 0 to 8 years. A similar 15N enrichment occurs in all Molluscs, Crustaceans, Insecta, Amphibia, Fish, Ave and Mammal species regardless of the difference in the form of excreted nitrogen and in laboratory cultured fish, brine shrimp and mice (+2.9 to +4.9%.). The excreted ammonia from guppy was sufficiently light to balance the concentration of 15N to animal body.

  18. Rapid, storm-induced changes in the natural abundance of 15N in a planktonic ecosystem, Chesapeake Bay, USA

    NASA Astrophysics Data System (ADS)

    Montoya, J. P.; Korrigan, S. G.; McCarthy, J. J.

    1991-12-01

    Samples of dissolved inorganic nitrogen (DIN), particulate nitrogen (PN) and two species of Zooplankton were collected during two north-south transects of the Chesapeake Bay in the autumn of 1984 (27-28 September and 3-5 October). During the first transect, the natural abundance of 15N ( δ15N) in the major dissolved and planktonic pools of nitrogen suggested that the δ15N of PN was largely determined by isotopic fractionation during uptake of NH 4+ by phytoplankton. Averaged over the transect as a whole, the δ15N of the herbivorous calanoid copepod Acartia tonsa was 4.1‰ higher than that of the PN, while the δ15N of the carnivorous ctenophore Mnemiopsis leidyi was 6.4‰ higher than that of the PN. In the interval between the two transects, storm-induced mixing of the water column resulted in the injection of NH 4+ into the surface layer of the bay. This perturbation in the estuarine nitrogen cycle resulted in marked changes in the δ15N of the major dissolved and planktonic pools of nitrogen in the bay. In combination with ancillary physical, chemical and biological data, these changes in δ15N provided estimates of the isotopic fractionation factor for NH 4+ uptake by phytoplankton ( α = 1.0065 -1.0080) as well as the turnover time of nitrogen in Acartia tonsa (6.0-9.6 days). Despite the changes in δ15N observed during this cruise, the relative distribution of 15N between trophic levels was preserved: during the second transect, the difference in δ15N between Acartia tonsa and PN was 3.6‰ and the difference in δ15N between Mnemiopsis leidyi and PN was 7.3‰. These results demonstrate that the natural abundance of 15N can change dramatically on a time scale of days and that time-series studies of the natural abundance of 15N can be a useful complement to studies using tracer additions of 15N to document nitrogen transformations in planktonic ecosystems.

  19. [Dynamics of 15N-isobutylidene diurea (IBDU) in sheep. 1. IBDU conversion in the digestive tract].

    PubMed

    Görsch, R; Bergner, H; Adam, K

    1978-07-01

    Four Merino Landrace wethers averaging 47.6 kg body weight were adapted to a semi-synthetic diet containing as the only N-source 60 g of IBDU per day. After the adaptation phase, on the 1 st experimental day the IBDU of the morning feed was given in 15N-labelled form (701 mg 15N-excess). After 2 1/2, 7 1/4, 12 and 24 hours the experimental animals were killed without having been fed again. The comparison of the IBDU-concentrations in the content of the rumen bottom with the residual rumen content did not allow to draw conclusions regarding IBDU-sedimentation at the bottom of the rumen. For the 15N-decline in the rumen content, a relationship was established following y = 76.3 - 2.62 (r = 0.96) (see fig. 2). In the order of killing times the following 15N-IBDU amounts were retrieved (% of intake): I = 15.6%, II = 24.1%, III = 3.3% and IV = 3.6%. 7 1/4 hours after starting the experiment, 40% of the 15N-labelled material were found in the rumen in the form IBDU; after 12 hours it came to 10%. Except for sheep I, 15N-urea was not found but in small amounts. Only sheep I and III revealed IBDU-traces in the abomasum, but in the small intestine of all sheep 2 to 6% of the amount taken in. This fact is explained with the endogenous influx of IBDU from the blood. An additional experimental sheep provided with a ligature at the abomasum entry, revealed that IBDU is absorbed from the rumen and allowed to enter the individual segments of the intestine in small amounts.

  20. 1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin.

    PubMed Central

    Campos-Olivas, R.; Bruix, M.; Santoro, J.; Martínez del Pozo, A.; Lacadena, J.; Gavilanes, J. G.; Rico, M.

    1996-01-01

    The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located. PMID:8732769

  1. Impact of Health Labels on Flavor Perception and Emotional Profiling: A Consumer Study on Cheese

    PubMed Central

    Schouteten, Joachim J.; De Steur, Hans; De Pelsmaeker, Sara; Lagast, Sofie; De Bourdeaudhuij, Ilse; Gellynck, Xavier

    2015-01-01

    The global increase of cardiovascular diseases is linked to the shift towards unbalanced diets with increasing salt and fat intake. This has led to a growing consumers’ interest in more balanced food products, which explains the growing number of health-related claims on food products (e.g., “low in salt” or “light”). Based on a within-subjects design, consumers (n = 129) evaluated the same cheese product with different labels. Participants rated liking, saltiness and fat flavor intensity before and after consuming four labeled cheeses. Even though the cheese products were identical, inclusion of health labels influenced consumer perceptions. Cheese with a “light” label had a lower overall expected and perceived liking compared to regular cheese. Although cheese with a “salt reduced” label had a lower expected liking compared to regular cheese, no lower liking was found when consumers actually consumed the labeled cheese. All labels also influenced the perceived intensities of the attributes related to these labels, e.g., for example salt intensity for reduced salt label. While emotional profiles of the labeled cheeses differed before tasting, little differences were found when actual tasting these cheeses. In conclusion, this study shows that health-related labels might influence the perceived flavor and emotional profiles of cheese products. PMID:26690211

  2. Effects of Four Different Restoration Treatments on the Natural Abundance of 15N Stable Isotopes in Plants

    PubMed Central

    Temperton, Vicky M.; Märtin, Lea L. A.; Röder, Daniela; Lücke, Andreas; Kiehl, Kathrin

    2012-01-01

    δ15N signals in plant and soil material integrate over a number of biogeochemical processes related to nitrogen (N) and therefore provide information on net effects of multiple processes on N dynamics. In general little is known in many grassland restoration projects on soil–plant N dynamics in relation to the restoration treatments. In particular, δ15N signals may be a useful tool to assess whether abiotic restoration treatments have produced the desired result. In this study we used the range of abiotic and biotic conditions provided by a restoration experiment to assess to whether the restoration treatments and/or plant functional identity and legume neighborhood affected plant δ15N signals. The restoration treatments consisted of hay transfer and topsoil removal, thus representing increasing restoration effort, from no restoration measures, through biotic manipulation to major abiotic manipulation. We measured δ15N and %N in six different plant species (two non-legumes and four legumes) across the restoration treatments. We found that restoration treatments were clearly reflected in δ15N of the non-legume species, with very depleted δ15N associated with low soil N, and our results suggest this may be linked to uptake of ammonium (rather than nitrate). The two non-legume species differed considerably in their δ15N signals, which may be related to the two species forming different kinds of mycorrhizal symbioses. Plant δ15N signals could clearly separate legumes from non-legumes, but our results did not allow for an assessment of legume neighborhood effects on non-legume δ15N signals. We discuss our results in the light of what the δ15N signals may be telling us about plant–soil N dynamics and their potential value as an indicator for N dynamics in restoration. PMID:22645597

  3. Effects of four different restoration treatments on the natural abundance of (15)n stable isotopes in plants.

    PubMed

    Temperton, Vicky M; Märtin, Lea L A; Röder, Daniela; Lücke, Andreas; Kiehl, Kathrin

    2012-01-01

    δ(15)N signals in plant and soil material integrate over a number of biogeochemical processes related to nitrogen (N) and therefore provide information on net effects of multiple processes on N dynamics. In general little is known in many grassland restoration projects on soil-plant N dynamics in relation to the restoration treatments. In particular, δ(15)N signals may be a useful tool to assess whether abiotic restoration treatments have produced the desired result. In this study we used the range of abiotic and biotic conditions provided by a restoration experiment to assess to whether the restoration treatments and/or plant functional identity and legume neighborhood affected plant δ(15)N signals. The restoration treatments consisted of hay transfer and topsoil removal, thus representing increasing restoration effort, from no restoration measures, through biotic manipulation to major abiotic manipulation. We measured δ(15)N and %N in six different plant species (two non-legumes and four legumes) across the restoration treatments. We found that restoration treatments were clearly reflected in δ(15)N of the non-legume species, with very depleted δ(15)N associated with low soil N, and our results suggest this may be linked to uptake of ammonium (rather than nitrate). The two non-legume species differed considerably in their δ(15)N signals, which may be related to the two species forming different kinds of mycorrhizal symbioses. Plant δ(15)N signals could clearly separate legumes from non-legumes, but our results did not allow for an assessment of legume neighborhood effects on non-legume δ(15)N signals. We discuss our results in the light of what the δ(15)N signals may be telling us about plant-soil N dynamics and their potential value as an indicator for N dynamics in restoration.

  4. The impact of nutritional labels and socioeconomic status on energy intake. An experimental field study.

    PubMed

    Crockett, Rachel A; Jebb, Susan A; Hankins, Matthew; Marteau, Theresa M

    2014-10-01

    There is some evidence for paradoxical effects of nutritional labelling on energy intake particularly amongst restrained eaters and those with a higher body mass index (BMI) resulting in greater consumption of energy from foods with a positive health message (e.g. "low-fat") compared with the same foods, unlabelled. This study aimed to investigate, in a UK general population sample, the likelihood of paradoxical effects of nutritional labelling on energy intake. Participants (n = 287) attended a London cinema and were offered a large tub of salted or toffee popcorn. Participants were randomised to receive their selected flavour with one of three labels: a green low-fat label, a red high-fat label or no label. Participants watched two film clips while completing measures of demographic characteristics, emotional state and taste of the popcorn. Following the experiment, popcorn consumption was measured. There were no main effects of nutritional labelling on consumption. Contrary to predictions neither BMI nor weight concern moderated the effect of label on consumption. There was a three-way interaction between low-fat label, weight concern and socioeconomic status (SES) such that weight-concerned participants of higher SES who saw a low-fat label consumed more than weight unconcerned participants of similar SES (t = -2.7, P = .04). By contrast, weight-concerned participants of lower SES seeing either type of label, consumed less than those seeing no label (t = -2.04, P = .04). Nutritional labelling may have different effects in different socioeconomic groups. Further studies are required to understand fully the possible contribution of food labelling to health inequalities. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Marking Drosophila suzukii (Diptera: Drosophilidae) With Rubidium or 15N.

    PubMed

    Klick, J; Yang, W Q; Bruck, D J

    2015-06-01

    Drosophila suzukii Matsumura (Diptera: Drosophilidae) has caused significant economic damage to berry and stone fruit production regions. Markers that are systemic in plants and easily transferred to target organisms are needed to track D. suzukii exploitation of host resources and trophic interactions. High and low concentrations of the trace element, rubidium (Rb), and the stable isotope, 15N, were tested to mark D. suzukii larvae feeding on fruits of enriched strawberry plants grown in containers under greenhouse conditions. Fly marker content and proportion of flies marked 1, 7, and 14 d after emergence from enriched fruits and fly dry mass were analyzed. Nearly 100% of the flies analyzed 14 d after emerging from 15N-enriched plants were marked, whereas only 30-75% and 0-3% were marked 14 d after emerging from high and low Rb concentration plants, respectively. Rapid Rb decay, strong 15N persistence, and the economics of using these markers in the field to elucidate D. suzukii pest ecology are discussed. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. 15N chemical shift referencing in solid state NMR.

    PubMed

    Bertani, Philippe; Raya, Jésus; Bechinger, Burkhard

    2014-01-01

    Solid-state NMR spectroscopy has much advanced during the last decade and provides a multitude of data that can be used for high-resolution structure determination of biomolecules, polymers, inorganic compounds or macromolecules. In some cases the chemical shift referencing has become a limiting factor to the precision of the structure calculations and we have therefore evaluated a number of methods used in proton-decoupled (15)N solid-state NMR spectroscopy. For (13)C solid-state NMR spectroscopy adamantane is generally accepted as an external standard, but to calibrate the (15)N chemical shift scale several standards are in use. As a consequence the published chemical shift values exhibit considerable differences (up to 22 ppm). In this paper we report the (15)N chemical shift of several commonly used references compounds in order to allow for comparison and recalibration of published data and future work. We show that (15)NH4Cl in its powdered form (at 39.3 ppm with respect to liquid NH3) is a suitable external reference as it produces narrow lines when compared to other reference compounds and at the same time allows for the set-up of cross-polarization NMR experiments. The compound is suitable to calibrate magic angle spinning and static NMR experiments. Finally the temperature variation of (15)NH4Cl chemical shift is reported.

  7. Cold brittleness of corrosion-resistant maraging steel 08Kh15N5D2T

    NASA Astrophysics Data System (ADS)

    Makhneva, T. M.

    2012-03-01

    Results of a study of the effect of the method of remelting and of heat treatment modes on the behavior of serial curves of impact toughness and on the position of cold-shortness threshold in steel 08Kh15N5D2T are presented.

  8. Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.

    PubMed

    Pandey, Manoj Kumar; Vivekanandan, Subramanian; Ahuja, Shivani; Pichumani, Kumar; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2012-06-21

    Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the (15)N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction, and the amino acid sequence, there is a need for accurately determined CSA tensors from proteins. In this study, we report the backbone amide-(15)N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit Cytochrome b(5) (cytb(5)), determined using the (15)N CSA/(15)N-(1)H dipolar transverse cross-correlation rates. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions are -187.9, -166.0, and -161.1 ppm, respectively, with an overall average value of -171.7 ppm. While the average CSA value determined from this study is in good agreement with previous solution NMR experiments on small globular proteins, the CSA value determined for residues in helical conformation is slightly larger, which may be attributed to the paramagnetic effect from Fe(III) of the heme unit in cytb(5). However, like in previous solution NMR studies, the CSA values reported in this study are larger than the values measured from solid-state NMR experiments. We believe that the CSA parameters reported in this study will be useful in determining the structure, dynamics, and orientation of proteins, including membrane proteins, using NMR spectroscopy.

  9. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    NASA Astrophysics Data System (ADS)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  10. Compound-specific δ15N and chlorin preservation in surface sediments of the Peru Margin with implications for ancient bulk δ15N records

    NASA Astrophysics Data System (ADS)

    Junium, Christopher K.; Arthur, Michael A.; Freeman, Katherine H.

    2015-07-01

    Understanding the processes that control the preservation of paleoceanographic proxies is of clear importance. Surface sediments from the Peru Margin oxygen-minimum zone are subject to lateral and downslope transport by bottom currents that decrease organic matter (OM) quality. Indicators of bulk OM quality (pyrolysis hydrogen index, pyrolysis S1 + S2 and C/N) demonstrate significant degradation between 150 and 400 m water depth, within the oxygen-minimum zone. Concentrations of the three most abundant chlorins (chlorophyllone, pheophytin and pyropheophytin) decrease from 750 to 150 nmol g TOC-1 from 150 to 400 m water depth though the relative abundances of the chlorins in an individual sample do not change. This suggests that the three chlorins have similar reactivity over the ambient conditions. Values for δ15N of bulk sediments (δ15Nbulk) decrease by 3‰ from the inner shelf to the upper slope (1000 m) but co-occurring compound-specific δ15N values (δ15Nchlorin) do not decrease downslope. The low variability of δ15Nchlorin values supports a single source for the chlorins, and demonstrates the recalcitrance of δ15Nchlorin values despite degradation. This set of observation raises questions about which type of OM fraction best records 'primary' signatures. We assess two possible models to guide our interpretation of these disparate datasets (1) that decreasing δ15Nbulk values are the result of degradation of a 15N-enriched fraction during downslope transport, and that δ15Nchlorin values reflect primary values; (2) that δ15Nbulk values are primary and that chlorins are derived from material transported from upslope. These data reaffirm that in active sedimentary environments such as the Eastern Tropical Pacific, transport of OM can significantly alter bulk geochemical parameters of OM integrity, but the impacts on the δ15N record of bulk sediments and chlorins are less clear, and require more study to be thoroughly understood.

  11. Rapid, storm-induced changes in the natural abundance of sup 15 N in a planktonic ecosystem, Chesapeake Bay, USA

    SciTech Connect

    Montoya, J.P.; McCarthy, J.J. ); Horrigan, S.G. )

    1991-12-01

    Samples of dissolved inorganic nitrogen (DIN), particulate nitrogen (PN), and two species of zooplankton were collected during two north-south transects of the Chesapeake Bay in the autumn of 1984 (27-28 September and 3-5 October). During the first transect, the natural abundance of {sup 15}N ({delta} {sup 15}N) in the major dissolved and planktonic pools of nitrogen suggested that the {delta}{sup 15}N of PN was largely determined by isotopic fractionation during uptake of NH{sub 4}{sup +} by phytoplankton. Averaged over the transect as a whole, the {delta}{sup 15}N of the herbivorous calanoid copepod Acartia tonsa was 4.1% higher than that of the PN, while the {delta}{sup 15}N of the carnivorous ctenophore Mnemiopsis leidyi was 6.4% higher than that of the PN. In the interval between the two transects, storm-induced mixing of the water column resulted in the injection of NH{sub 4}{sup +} into the surface layer of the bay. In combination with ancillary physical, chemical, and biological data, these changes in {delta}{sup 15}N provided estimates of the isotopic fractionation factor for NH{sub 4}{sup +} uptake by phytoplankton ({alpha} = 1.0065-1.0080) as well as the turnover time of nitrogen in Acartia tonsa (6.0-9.6 days). Despite the changes in {delta}{sup 15}N observed during this cruise, the relative distribution of {sup 15}N between trophic levels was preserved: during the second transect, the difference in {delta}{sup 15}N between Acartia tonsa and PN was 3.6%, and the difference in {delta}{sup 15}N between Mnemiopsis leidyi and PN was 7.3%. These results demonstrate that the natural abundance of {sup 15}N can change dramatically on a time scale of days, and that time-series studies of the natural abundance of {sup 15}N can be a useful complement to studies using tracer additions of {sup 15}N to document nitrogen transformations in planktonic ecosystems.

  12. IOM Review of FDA--approved biologics labeled or studied for pediatric use.

    PubMed

    Field, Marilyn J; Ellinger, Lara K; Boat, Thomas F

    2013-02-01

    Studies have examined the extent to which public policies such as the Best Pharmaceuticals for Children Act have increased pediatric information in drug labeling. Little attention has focused on pediatric labeling of biologics. This analysis examines the extent to which biologics are labeled for pediatric use or have been studied in children. The analysis covers the 96 biologics (excluding vaccines) that were first licensed by the Food and Drug Administration between 1997 and 2010 and were still marketed as of 2010. Product labeling was consulted for information on approved pediatric uses, pediatric studies, or pediatric safety warnings based on analyses of adverse events. The online database ClinicalTrials.gov was searched for registered pediatric studies of these biologics. A separate analysis examined labeling and studies for 55 vaccines. For ∼60% of the 96 biologics, labeling shows approved pediatric use or pediatric study information or both. Approximately 85% of the biologics have ≥1 registered pediatric trial completed, underway, or planned. Overall, ∼90% are labeled for pediatric use, have pediatric information in the label, have a registered pediatric study, or have some combination of these characteristics. For the 55 analyzed vaccines, the corresponding figure is 95%. A majority of biologics approved in the past 15 years include some pediatric information in their labeling, and pediatric trials have been registered for a substantial majority of these products.

  13. Species differences in nitrogen cycling in a humid sub-tropical forest inferred from 15N natural abundance

    NASA Astrophysics Data System (ADS)

    Abdisa Gurmesa, Geshere; Lu, Xiankai; Gundersen, Per; Mao, Qinggong; Zhou, Kaijun; Mo, Jiangming

    2017-04-01

    Studies of natural abundance of stable nitrogen isotope (δ15N) of ecosystems can provide integrated information about N status and N cycling rates within the ecosystems. Plant species with different N cycling traits can affect ecosystem δ15N, but such differences are poorly explored in tropical forests. This study evaluates the extent of variation in plant δ15N among co-occurring sub-tropical tropical tree species in old-growth mixed broadleaved forest in southern China. We compared leaf δ15N values among five co-occurring tree species under ambient deposition (control plots), and variation in plant δ15N response to a decade of N addition (N-plots) and to a one-year enriched 15N addition to both treatments in the study forest. We found significant differences in leaf δ15N values among tree species (up to 3‰) both in control and N-plots. Responses of leaf δ15N to N and 15N addition also differ among the tree species. These differences are explained by differences in N acquisition strategies (dependence on soil N and/or deposition N among the plant species) that is partly related to differences in mycorrhizal association among the studied plants. Our results indicate that plant species in N-rich tropical forests could have distinct N cycling traits as observed in many predominantly N-limited temperate and boreal forests. The finding, therefore, highlights the importance of considering tree species variation in studying N cycling in N-rich tropical forests.

  14. Follow the Carbon: Isotopic Labeling Studies of Early Earth Aerosol

    NASA Astrophysics Data System (ADS)

    Hicks, Raea K.; Day, Douglas A.; Jimenez, Jose L.; Tolbert, Margaret A.

    2016-11-01

    Despite the faint young Sun, early Earth might have been kept warm by an atmosphere containing the greenhouse gases CH4 and CO2 in mixing ratios higher than those found on Earth today. Laboratory and modeling studies suggest that an atmosphere containing these trace gases could lead to the formation of organic aerosol haze due to UV photochemistry. Chemical mechanisms proposed to explain haze formation rely on CH4 as the source of carbon and treat CO2 as a source of oxygen only, but this has not previously been verified experimentally. In the present work, we use isotopically labeled precursor gases and unit-mass resolution (UMR) and high-resolution (HR) aerosol mass spectrometry to examine the sources of carbon and oxygen to photochemical aerosol formed in a CH4/CO2/N2 atmosphere. UMR results suggest that CH4 contributes 70-100% of carbon in the aerosol, while HR results constrain the value from 94% to 100%. We also confirm that CO2 contributes approximately 10% of the total mass to the aerosol as oxygen. These results have implications for the geochemical interpretations of inclusions found in Archean rocks on Earth and for the astrobiological potential of other planetary atmospheres.

  15. Follow the Carbon: Isotopic Labeling Studies of Early Earth Aerosol.

    PubMed

    Hicks, Raea K; Day, Douglas A; Jimenez, Jose L; Tolbert, Margaret A

    2016-11-01

    Despite the faint young Sun, early Earth might have been kept warm by an atmosphere containing the greenhouse gases CH4 and CO2 in mixing ratios higher than those found on Earth today. Laboratory and modeling studies suggest that an atmosphere containing these trace gases could lead to the formation of organic aerosol haze due to UV photochemistry. Chemical mechanisms proposed to explain haze formation rely on CH4 as the source of carbon and treat CO2 as a source of oxygen only, but this has not previously been verified experimentally. In the present work, we use isotopically labeled precursor gases and unit-mass resolution (UMR) and high-resolution (HR) aerosol mass spectrometry to examine the sources of carbon and oxygen to photochemical aerosol formed in a CH4/CO2/N2 atmosphere. UMR results suggest that CH4 contributes 70-100% of carbon in the aerosol, while HR results constrain the value from 94% to 100%. We also confirm that CO2 contributes approximately 10% of the total mass to the aerosol as oxygen. These results have implications for the geochemical interpretations of inclusions found in Archean rocks on Earth and for the astrobiological potential of other planetary atmospheres. Key Words: Atmosphere-Early Earth-Planetary atmospheres-Carbon dioxide-Methane. Astrobiology 16, 822-830.

  16. Influence of 15N enrichment on the net isotopic fractionation factor during the reduction of nitrate to nitrous oxide in soil.

    PubMed

    Mathieu, Olivier; Lévêque, Jean; Hénault, Catherine; Ambus, Per; Milloux, Marie-Jeanne; Andreux, Francis

    2007-01-01

    Nitrous oxide, a greenhouse gas, is mainly emitted from soils during the denitrification process. Nitrogen stable-isotope investigations can help to characterise the N(2)O source and N(2)O production mechanisms. The stable-isotope approach is increasingly used with (15)N natural abundance or relatively low (15)N enrichment levels and requires a good knowledge of the isotopic fractionation effect inherent to this biological mechanism. This paper reports the measurement of the net and instantaneous isotopic fractionation factor (alpha(s/p) (i)) during the denitrification of NO(3) (-) to N(2)O over a range of (15)N substrate enrichments (0.37 to 1.00 atom% (15)N). At natural abundance level, the isotopic fractionation effect reported falls well within the range of data previously observed. For (15)N-enriched substrate, the value of alpha(s/p) (i) was not constant and decreased from 1.024 to 1.013, as a direct function of the isotopic enrichment of the labelled nitrate added. However, for enrichment greater than 0.6 atom% (15)N, the value of alpha(s/p) (i) seems to be independent of substrate isotopic enrichment. These results suggest that for isotopic experiments applied to N(2)O emissions, the use of low (15)N-enriched tracers around 1.00 atom% (15)N is valid. At this enrichment level, the isotopic effect appears negligible in comparison with the enrichment of the substrate. Copyright (c) 2007 John Wiley & Sons, Ltd.

  17. Measuring and Modeling Highly Accurate (15) N Chemical Shift Tensors in a Peptide.

    PubMed

    Soss, Sarah E; Flynn, Peter F; Iuliucci, Robbie J; Young, Robert P; Mueller, Leonard J; Hartman, Joshua; Beran, Gregory J O; Harper, James K

    2017-08-18

    NMR studies measuring chemical shift tensors are increasingly being employed to assign structure in difficult-to-crystallize solids. For small organic molecules, such studies usually focus on (13) C sites, but proteins and peptides are more commonly described using (15) N amide sites. An important and often neglected consideration when measuring shift tensors is the evaluation of their accuracy against benchmark standards, where available. Here we measure (15) N tensors in the dipeptide glycylglycine at natural abundance using the slow-spinning FIREMAT method with SPINAL-64 decoupling. The accuracy of these (15) N tensors is evaluated by comparing to benchmark single crystal NMR (15) N measurements and found to be statistically indistinguishable. These FIREMAT experimental results are further used to evaluate the accuracy of theoretical predictions of tensors from four different density functional theory (DFT) methods that include lattice effects. The best theoretical approach provides a root mean square (rms) difference of ±3.9 ppm and is obtained from a fragment-based method and the PBE0 density functional. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. NMR study of non-structural proteins-part III: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Chikungunya virus (CHIKV).

    PubMed

    Lykouras, Michail V; Tsika, Aikaterini C; Lichière, Julie; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2017-09-05

    Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130-190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.

  19. A spin label study of conformational changes in cytochrome c.

    PubMed

    Postnikova, G B; Gorbunova, N P; Volkenstein, M V

    1983-04-01

    Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.

  20. Organically treated biochar increases plant production and reduces N2O emissions: mechanistic insights by 15N tracing

    NASA Astrophysics Data System (ADS)

    Kammann, Claudia; Messerschmidt, Nicole; Clough, Tim; Schmidt, Hans-Peter; Marhan, Sven; Koyro, Hans-Werner; Steffens, Diedrich; Müller, Christoph

    2014-05-01

    Pyrogenic carbon (biochar) offers considerable potential for carbon capture and soil storage (CCSS) compared to other, less recalcitrant soil-C additives. Recent meta-analysis demonstrated that it can significantly reduce agricultural N2O emissions. Freshly produced biochars, however, do not always have yield-improving effects, i.e. there is no immediate economic incentive for using it. Hence, combining biochar with organic nutrient-rich amendments may be a promising agricultural strategy to accelerate CCSS, but it is unclear if biochar still reduces N2O emissions, in particular when it may act as nutrient carrier. We explored the potential of biochar to improve the GHG-cost/yield ratio and thereby its socio-economic value as soil amendment in two subsequent studies under controlled conditions: (1) A proof-of-concept study where the effects of untreated biochar were compared to those of co-composted biochar combined with stepwise improved nutritional regimes (+/- compost; +/- mineral-N application), and (2) a 15N-labeling-tracing study to unravel N exchange on biochar particles and N2O production and reduction mechanisms. Both studies were carried out in nutrient-poor sandy soils, the most likely initial target soils for biochar-CCSS strategies. While the untreated biochar reduced plant growth under N-limiting conditions, or at best did not reduce it, the co-composted biochar always significantly stimulated plant growth. The relative stimulation was largest with the lowest nutrient additions (305% versus 61% of control with untreated biochar). Electro-ultra-filtration analyses revealed that the co-composted but not the untreated biochar carried considerable amounts of easily extractable as well as more strongly sorbed plant nutrients, in particular nitrate and phosphorus. The subsequent 15N labelling-tracing study revealed that the co-composted biochar still (i) acted as a mineral-N exchange site for nitrate and ammonium despite its N-preloading, (ii) reduced N2O

  1. Cereal grain, rachis and pulse seed amino acid δ15N values as indicators of plant nitrogen metabolism.

    PubMed

    Styring, Amy K; Fraser, Rebecca A; Bogaard, Amy; Evershed, Richard P

    2014-01-01

    Natural abundance δ(15)N values of plant tissue amino acids (AAs) reflect the cycling of N into and within plants, providing an opportunity to better understand environmental and anthropogenic effects on plant metabolism. In this study, the AA δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. It was found that the δ(15)N values of cereal grain and rachis AAs could be largely attributed to metabolic pathways involved in their biosynthesis and catabolism. The relative (15)N-enrichment of phenylalanine can be attributed to its involvement in the phenylpropanoid pathway and glutamate has a δ(15)N value which is an average of the other AAs due to its central role in AA-N cycling. The relative AA δ(15)N values of broad bean and pea seeds were very different from one another, providing evidence for differences in the metabolic routing of AAs to the developing seeds in these leguminous plants. This study has shown that AA δ(15)N values relate to known AA biosynthetic pathways in plants and thus have the potential to aid understanding of how various external factors, such as source of assimilated N, influence metabolic cycling of N within plants.

  2. Experimental study of super paramagnetic iron oxide labeled synovial mesenchymal stem cells.

    PubMed

    Yu, Fang-Yuan; Li, Hong-Hang; Chen, Chang-Hui; Bi, Sheng-Rong

    2015-01-01

    To investigate the feasibility and changes of biological characteristics before and after synovial mesenchymal stem cells (SMSCs) labelled by super paramagnetic iron oxide (SPIO). The rabbit SMSCs were isolated, cultured, purified and identified in vitro. After adding the different concentrations of SPIO-labelled liquid, the cells were incubated 24 h in 37°C carbon dioxide incubator. The labeled-cell samples were observed by Prussian blue staining, transmission electron microscope (TEM) and the cell biology before and after the labeling was compared. The blue stained particles could be seen in the cytoplasm; the SPIO label was positive in 95% SMSC cells. With the concentration of the label liquid increasing, the blue-stained cytoplasm became darker. A large number of high electron density particles could be seen in the cytoplasm and in the pinocytosis vesicles by TEM, which suggested SPIO label positive. When the SPIO concentration was (12.5~50) μg/mL, the differences in cell proliferation and cell viability between the SMSCs after labelling and the SMSCs before labelling were not significant; when the concentration was over 100 μg/mL, the cell proliferation and cell viability were inhibited. A certain concentration range of SPIO can safely label the rabbit SMSC according to this study, which is important for solving the problem of tracing SMSCs in the joints.

  3. Predicting the denitrification capacity of sandy aquifers from in situ measurements using push-pull 15N tracer tests

    NASA Astrophysics Data System (ADS)

    Eschenbach, W.; Well, R.; Walther, W.

    2015-04-01

    Knowledge about the spatial variability of in situ denitrification rates (Dr(in situ)) and their relation to the denitrification capacity in nitrate-contaminated aquifers is crucial to predict the development of groundwater quality. Therefore, 28 push-pull 15N tracer tests for the measurement of in situ denitrification rates were conducted in two sandy Pleistocene aquifers in northern Germany. The 15N analysis of denitrification-derived 15N-labelled N2 and N2O dissolved in water samples collected during the push-pull 15N tracer tests was performed using isotope ratio mass spectrometry (IRMS) in the lab and additionally for some tracer tests online in the field with a quadrupole membrane inlet mass spectrometer (MIMS) in order to test the feasibility of on-site real-time 15N analysis. Aquifer material from the same locations and depths as the push-pull injection points was incubated, and the initial and cumulative denitrification after 1 year of incubation (Dcum(365)) as well as the stock of reduced compounds (SRC) was compared with in situ measurements of denitrification. This was done to derive transfer functions suitable to predict Dcum(365) and SRC from Dr(in situ). Dr(in situ) ranged from 0 to 51.5 μg N kg-1 d-1. Denitrification rates derived from on-site isotope analysis using MIMS satisfactorily coincided with laboratory analysis by conventional IRMS, thus proving the feasibility of in situ analysis. Dr(in situ) was significantly higher in the sulfidic zone of both aquifers compared to the zone of non-sulfidic aquifer material. Overall, regressions between the Dcum(365) and SRC of the tested aquifer material with Dr(in situ) exhibited only a modest linear correlation for the full data set. However, the predictability of Dcum(365) and SRC from Dr(in situ) data clearly increased for aquifer samples from the zone of NO3--bearing groundwater. In the NO3--free aquifer zone, a lag phase of denitrification after NO3- injections was observed, which confounded the

  4. Predicting the denitrification capacity of sandy aquifers from in situ measurements using push-pull 15N tracer tests

    NASA Astrophysics Data System (ADS)

    Eschenbach, W.; Well, R.; Walther, W.

    2014-12-01

    Knowledge about the spatial variability of in situ denitrification rates (Dr(in situ)) and their relation to the denitrification capacity in nitrate-contaminated aquifers is crucial to predict the development of groundwater quality. Therefore, 28 push-pull 15N tracer tests for the measurement of in situ denitrification rates were conducted in two sandy Pleistocene aquifers in Northern Germany. The 15N analysis of denitrification derived 15N labelled N2 and N2O dissolved in water samples collected during the push-pull 15N tracer tests was performed by isotope ratio mass spectrometry (IRMS) in the lab and additionally for some tracer tests online in the field with a quadrupole membrane inlet mass spectrometer (MIMS), in order to test the feasibility of on-site real-time 15N analysis. Aquifer material from the same locations and depths as the push-pull injection points was incubated and the initial and cumulative denitrification after one year of incubation (Dcum(365)) as well as the stock of reduced compounds (SRC) was compared with in situ measurements of denitrification. This was done to derive transfer functions suitable to predict Dcum(365) and SRC from Dr(in situ). Dr(in situ) ranged from 0 to 51.5 μg N kg-1 d-1. Denitrification rates derived from on-site isotope analysis using membrane-inlet mass spectrometry satisfactorily coincided with laboratory analysis by conventional isotope ratio mass spectrometry, thus proving the feasibility of in situ analysis. Dr(in situ) was significantly higher in the sulphidic zone of both aquifers compared to the zone of non-sulphidic aquifer material. Overall, regressions between the Dcum(365) and SRC of the tested aquifer material with Dr(in situ) exhibited only a modest linear correlation for the full data set. But the predictability of Dcum(365) and SRC from Dr(in situ) data clearly increased for aquifer samples from the zone of NO3--bearing groundwater. In the NO3--free aquifer zone a lag phase of denitrification after NO3

  5. δ15N as a proxy for historic anthropogenic nitrogen loading in Charleston Harbor, SC, USA

    NASA Astrophysics Data System (ADS)

    Payne, T. N.; Andrus, C. F. T.

    2015-12-01

    Bivalve shell geochemistry can serve as a useful indicator of changes in coastal environments. There is increasing interest in developing paleoenvironmental proxies from mollusk shell organic components. Numerous studies have focused on how the δ15N obtained from bivalve tissues can be used to trace present-day wastewater input into estuaries. However, comparatively little attention has been paid to tracing the impact of anthropogenic nitrogen loading into estuaries over time. By measuring historic levels of δ15N in the organic fraction of oyster shells (Crassostrea virginica) from archaeological sites around Charleston Harbor and comparing those levels to the δ15N content of modern shells, it is possible to assess how nitrogen has fluctuated historically in the area. Whole-shell samples from the Late Archaic Period (~3000-4000 BP, Late Woodland Period (~1400-800 BP), 18th and 19th centuries, and modern controls were measured for %N and d15N. Evidence of increased anthropogenic input of N is expected to begin in the early historic period based on similar analysis in Chesapeake Bay. More ancient samples may give insight into baseline conditions prior to recent population growth and industrialization. This information could help understand how large-scale anthropogenic nitrogen loading has affected coastal ecosystems over time and guide future remediation. Furthermore, this project will help refine and improve this novel proxy of past environmental conditions.

  6. Plant and soil natural abundance delta (15)N: indicators of relative rates of nitrogen cycling in temperate forest ecosystems.

    PubMed

    Templer, Pamela H; Arthur, Mary A; Lovett, Gary M; Weathers, Kathleen C

    2007-08-01

    Watersheds within the Catskill Mountains, New York, receive among the highest rates of nitrogen (N) deposition in the northeastern United States and are beginning to show signs of N saturation. Despite similar amounts of N deposition across watersheds within the Catskill Mountains, rates of soil N cycling and N retention vary significantly among stands of different tree species. We examined the potential use of delta (15)N of plants and soils as an indicator of relative forest soil N cycling rates. We analyzed the delta (15)N of foliage, litterfall, bole wood, surface litter layer, fine roots and organic soil from single-species stands of American beech (Fagus grandifolia), eastern hemlock (Tsuga canadensis), red oak (Quercus rubra), and sugar maple (Acer saccharum). Fine root and organic soil delta (15)N values were highest within sugar maple stands, which correlated significantly with higher rates of net mineralization and nitrification. Results from this study suggest that fine root and organic soil delta (15)N can be used as an indicator of relative rates of soil N cycling. Although not statistically significant, delta (15)N was highest within foliage, wood and litterfall of beech stands, a tree species associated with intermediate levels of soil N cycling rates and forest N retention. Our results show that belowground delta (15)N values are a better indicator of relative rates of soil N cycling than are aboveground delta (15)N values.

  7. δ15N-Size Relationships in River Invertebrate Communities: An Integrated Measure of Food web Structure?

    NASA Astrophysics Data System (ADS)

    Anderson, C.; Cabana, G.

    2005-05-01

    Anthropogenic perturbations can alter the composition and structure of benthic communities in rivers. Here, we examine the use of estimates of fish trophic position measured with stable nitrogen isotope ratios (δ15N), and δ15N-size relationships for the invertebrate community, as indicators of food web structure in rivers affected by various anthropogenic disturbances. Results revealed that δ15N-size relationships of invertebrate communities showed a variety of responses, some sites (50%) showing significant increases in δ15N with invertebrate size, whereas others did not. Similarly, the number of trophic levels between invertebrate-eating fish and primary consumers varied greatly among study sites, ranging from 0.6 to 2.2 levels. To investigate whether these estimates of fish trophic position integrated changes in invertebrate community structure, we compared slopes of invertebrate δ15N versus size with slopes of baseline-corrected fish δ15N versus size. Both slopes tended to be correlated, although the relationship was not significant (r2 = 0.26, p = 0.07). This trend suggests, however, that invertebrate-feeding fish trophic position measured with δ15N might reflect overall community structure, and could possibly be used as an easily measured indicator of food chain collapse following anthropogenic perturbations.

  8. The degree of urbanization across the globe is not reflected in the δ(15)N of seagrass leaves.

    PubMed

    Christiaen, Bart; Bernard, Rebecca J; Mortazavi, Behzad; Cebrian, Just; Ortmann, Alice C

    2014-06-30

    Many studies show that seagrass δ(15)N ratios increase with the amount of urbanization in coastal watersheds. However, there is little information on the relationship between urbanization and seagrass δ(15)N ratios on a global scale. We performed a meta-analysis on seagrass samples from 79 independent locations to test if seagrass δ(15)N ratios correlate with patterns of population density and fertilizer use within a radius of 10-200 km around the sample locations. Our results show that seagrass δ(15)N ratios are more influenced by intergeneric and latitudinal differences than the degree of urbanization or the amount of fertilizer used in nearby watersheds. The positive correlation between seagrass δ(15)N ratios and latitude hints at an underlying pattern in discrimination or a latitudinal gradient in the (15)N isotopic signature of nitrogen assimilated by the plants. The actual mechanisms responsible for the correlation between δ(15)N and latitude remain unknown. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Vertical δ13C and δ15N changes during pedogenesis

    NASA Astrophysics Data System (ADS)

    Brunn, Melanie; Spielvogel, Sandra; Wells, Andrew; Condron, Leo; Oelmann, Yvonne

    2015-04-01

    The natural abundance of soil organic matter (SOM) stable C and N isotope ratios are subjected to vertical changes throughout the soil profile. This vertical distribution is a widely reported phenomenon across varieties of ecosystems and constitutes important insights of soil carbon cycling. In most ecosystems, SOM becomes enriched in heavy isotopes by several per mill in the first few centimeters of the topsoil. The enrichment of 13C in SOM with soil depth is attributed to biological and physical-chemical processes in soil e.g., plant physiological impacts, microbial decomposition, sorption and transport processes. Such vertical trends in 13C and 15N abundance have rarely been related to SOM composition during pedogenesis. The aims of our study were to investigate short and long-term δ13C and δ15N depth changes and their interrelations under progressing pedogenesis and ecosystem development. We sampled soils across the well studied fordune progradation Haast-chronosequence, a dune ridge system under super-humid climate at the West Coast of New Zealand's South Island (43° 53' S, 169° 3' E). Soils from 11 sites with five replicates each covered a time span of around 2870 yr of soil development (from Arenosol to Podzol). Vertical changes of δ13C and δ15N values of SOM were investigated in the organic layers and in 1-cm depth intervals of the upper 10 cm of the mineral soil. With increasing soil depth SOM became enriched in δ13C by 1.9 ± SE 0.1 o and in δ15N by 6.0 ± 0.4 ‰˙Litter δ13C values slightly decreased with increasing soil age (r = -0.61; p = 0.00) likely due to less efficient assimilation linked to nutrient limitations. Fractionation processes during mycorrhizal transfer appeared to affect δ15N values in the litter. We found a strong decrease of δ15N in the early succession stages ≤ 300 yr B.P. (r = -0.95; p = 0.00). Positive relations of vertical 13C and 15N enrichment with soil age might be related to decomposition and appeared to be

  10. Localization of 15N uptake in a Tibetan alpine Kobresia pasture

    NASA Astrophysics Data System (ADS)

    Schleuß, Per-Marten; Kuzyakov, Yakov

    2014-05-01

    The Kobresia Pygmea ecotone covers approximately 450.000 km2 and is of large global and regional importance due several socio-ecological aspects. For instance Kobresia pastures store high amounts of carbon, nitrogen and other nutrients, represent large grazing areas for herbivores, provide a fast regrowth after grazing events and protect against mechanical degradation and soil erosion. However, Kobresia pastures are assumed to be a grazing induced and are accompanied with distinct root mats varying in thickness between 5-30 cm. Yet, less is known about the morphology and the functions of this root mats, especially in the background of a progressing degradation due to changes of climate and management. Thus we aimed to identify the importance of single soil layers for plant nutrition. Accordingly, nitrogen uptake from different soil depths and its remain in above-ground biomass (AGB), belowground biomass (BGB) and soil were determined by using a 15N pulse labeling approach during the vegetation period in summer 2012. 15N urea was injected into six different soil depths (0.5 cm, 2.5 cm, 7.5 cm, 12.5 cm, 17.5 cm, 22.5 cm / for each 4 replicates) and plots were sampled 45 days after the labeling. For soil and BGB samples were taken in strict sample intervals of 0-1 cm, 1-5 cm, 5-10 cm, 10-15 cm, 15-20 cm, 20-25 cm. Results indicate that total recovery (including AGB, BGB and soil) was highest, if tracer was injected into the top 5 cm and subsequently decreased with decreasing injection depth. This is especially the case for the 15N recovery of BGB, which is clearly attributed to the root density and strongly decreased with soil depth. In contrast, the root activity derived from the 15N content of roots increased with soil depth, which is primary associated to a proportionate increase of living roots related to dead roots. However, most 15N was captured in plant biomass (67.5-85.3 % of total recovery), indicating high 15N uptake efficiency possibly due to N limitation

  11. Tracking the flow of bacterially derived 13C and 15N through soil faunal feeding channels.

    PubMed

    Crotty, F V; Blackshaw, R P; Murray, P J

    2011-06-15

    The soil food web has been referred to as a 'black box', a 'poor man's tropical rainforest' and an 'enigma', due to its opacity, diversity and the limited insight into feeding specificity. Here we investigate the flow of C and N through the soil food web as a way to gain understanding of the feeding interactions occurring. A bacterium, Pseudomonas lurida, was introduced to soil cores from two different habitats, a grassland and a woodland with the same soil type, enriched to 99 atom% in (13)C and (15)N, to trace the flow of bacterial C and N through the soil food web. Throughout the experiment the soil remained enriched in (13)C and (15)N. Almost all the invertebrates tested gained C and N enrichment indicative of the labelled bacteria, implying that bacterial feeding is a common mechanism within the soil. Only three groups were significantly enriched in both (13)C and (15)N in both habitats. These were Collembola (Entomobryomorpha), Acari (Oribatida), and Nematoda, indicating that these organisms are consuming the most bacteria within both systems. When the invertebrates were grouped into hypothesised trophic levels, those considered secondary decomposers were gaining the most enrichment across all invertebrates tested. This enrichment was also high in the micro-predators within the soil, implying that their main food source was the secondary decomposers, particularly the Collembola. Using an enriched bacterium to track the trophic transfer between organisms within the soil food web is a novel way of empirically showing that interactions are occurring, which normally cannot be seen.

  12. Foliar δ15N is affected by foliar nitrogen uptake, soil nitrogen, and mycorrhizae along a nitrogen deposition gradient.

    PubMed

    Vallano, Dena M; Sparks, Jed P

    2013-05-01

    Foliar nitrogen isotope (δ(15)N) composition patterns have been linked to soil N, mycorrhizal fractionation, and within-plant fractionations. However, few studies have examined the potential importance of the direct foliar uptake of gaseous reactive N on foliar δ(15)N. Using an experimental set-up in which the rate of mycorrhizal infection was reduced using a fungicide, we examined the influence of mycorrhizae on foliar δ(15)N in potted red maple (Acer rubrum) seedlings along a regional N deposition gradient in New York State. Mycorrhizal associations altered foliar δ(15)N values in red maple seedlings from 0.06 to 0.74 ‰ across sites. At the same sites, we explored the predictive roles of direct foliar N uptake, soil δ(15)N, and mycorrhizae on foliar δ(15)N in adult stands of A. rubrum, American beech (Fagus grandifolia), black birch (Betula lenta), and red oak (Quercus rubra). Multiple regression analysis indicated that ambient atmospheric nitrogen dioxide (NO2) concentration explained 0, 69, 23, and 45 % of the variation in foliar δ(15)N in American beech, red maple, red oak, and black birch, respectively, after accounting for the influence of soil δ(15)N. There was no correlation between foliar δ(13)C and foliar %N with increasing atmospheric NO2 concentration in most species. Our findings suggest that total canopy uptake, and likely direct foliar N uptake, of pollution-derived atmospheric N deposition may significantly impact foliar δ(15)N in several dominant species occurring in temperate forest ecosystems.

  13. Discrimination against 15N among recombinant inbred lines of Phaseolus vulgaris L. contrasting in phosphorus use efficiency for nitrogen fixation.

    PubMed

    Lazali, Mohamed; Bargaz, Adnane; Carlsson, Georg; Ounane, Sidi Mohamed; Drevon, Jean Jacques

    2014-02-15

    Although isotopic discrimination processes during nitrogen (N) transformations influence the outcome of (15)N based quantification of N2 fixation in legumes, little attention has been given to the effects of genotypic variability and environmental constraints such as phosphorus (P) deficiency, on discrimination against (15)N during N2 fixation. In this study, six Phaseolus vulgaris recombinant inbred lines (RILs), i.e. RILs 115, 104, 34 (P deficiency tolerant) and 147, 83, 70 (P deficiency sensitive), were inoculated with Rhizobium tropici CIAT899, and hydroaeroponically grown with P-sufficient (250 μmol P plant(-1) week(-1)) versus P-deficient (75 μmol P plant(-1) week(-1)) supply. Two harvests were done at 15 (before nodule functioning) and 42 (flowering stage) days after transplanting. Nodulation, plant biomass, P and N contents, and the ratios of (15)N over total N content ((15)N/Nt) for shoots, roots and nodules were determined. The results showed lower (15)N/Nt in shoots than in roots, both being much lower than in nodules. P deficiency caused a larger decrease in (15)N/Nt in shoots (-0.18%) than in nodules (-0.11%) for all of the genotypes, and the decrease in shoots was greatest for RILs 34 (-0.33%) and 104 (-0.25%). Nodule (15)N/Nt was significantly related to both the quantity of N2 fixed (R(2)=0.96***) and the P content of nodules (R(2)=0.66*). We conclude that the discrimination against (15)N in the legume N2-fixing symbiosis of common bean with R. tropici CIAT899 is affected by P nutrition and plant genotype, and that the (15)N/Nt in nodules may be used to screen for genotypic variation in P use efficiency for N2 fixation.

  14. Refining Prescription Warning Labels Using Patient Feedback: A Qualitative Study.

    PubMed

    Shiyanbola, Olayinka O; Smith, Paul D; Mansukhani, Sonal Ghura; Huang, Yen-Ming

    2016-01-01

    The complexity of written medication information hinders patients' understanding and leads to patient misuse of prescribed medications. Incorporating patient feedback in designing prescription warning labels (PWLs) is crucial in enhancing patient comprehension of medication warning instructions. This qualitative study explored patient feedback on five newly designed PWLs. In-depth semi-structured face-to-face interviews were conducted with 21 patients, who were 18 years and older, spoke English, and took a prescription medication. These patients were shown different variations of the five most commonly used PWLs-Take with Food, Do not Drink Alcohol, Take with a Full glass of Water, Do not Chew or Break, and Protect from Sunlight. The 60-minute interviews explored feedback on patient comprehension of the PWL instructions and their suggestions for improving the clarity of the PWLs. At the end of the interview, patient self-reported socio-demographic information was collected with a 3-minute survey and a brief health literacy assessment was completed using the Newest Vital Sign. Twenty-one patients completed the interviews. Most patients were female (n = 15, 71.4%) with ages ranging from 23 to 66 years old (mean: 47.6 ± 13.3). The mean health literacy score was 2.4 on a scale of 0-6. Qualitative content analysis based on the text, pictures, and placement of the PWLs on the pill bottle showed preferences for including 'WARNING' on the PWL to create alertness, inclusion of a picture together with the text, yellow color highlighting behind the text, and placement of the PWL on the front of the pill bottle. Although patients had positive opinions of the redesigned PWLs, patients wanted further improvements to the content and design of the PWLs for enhanced clarity and understandability.

  15. Refining Prescription Warning Labels Using Patient Feedback: A Qualitative Study

    PubMed Central

    Mansukhani, Sonal Ghura; Huang, Yen-Ming

    2016-01-01

    The complexity of written medication information hinders patients’ understanding and leads to patient misuse of prescribed medications. Incorporating patient feedback in designing prescription warning labels (PWLs) is crucial in enhancing patient comprehension of medication warning instructions. This qualitative study explored patient feedback on five newly designed PWLs. In-depth semi-structured face-to-face interviews were conducted with 21 patients, who were 18 years and older, spoke English, and took a prescription medication. These patients were shown different variations of the five most commonly used PWLs-Take with Food, Do not Drink Alcohol, Take with a Full glass of Water, Do not Chew or Break, and Protect from Sunlight. The 60-minute interviews explored feedback on patient comprehension of the PWL instructions and their suggestions for improving the clarity of the PWLs. At the end of the interview, patient self-reported socio-demographic information was collected with a 3-minute survey and a brief health literacy assessment was completed using the Newest Vital Sign. Twenty-one patients completed the interviews. Most patients were female (n = 15, 71.4%) with ages ranging from 23 to 66 years old (mean: 47.6 ± 13.3). The mean health literacy score was 2.4 on a scale of 0–6. Qualitative content analysis based on the text, pictures, and placement of the PWLs on the pill bottle showed preferences for including ‘WARNING’ on the PWL to create alertness, inclusion of a picture together with the text, yellow color highlighting behind the text, and placement of the PWL on the front of the pill bottle. Although patients had positive opinions of the redesigned PWLs, patients wanted further improvements to the content and design of the PWLs for enhanced clarity and understandability. PMID:27258026

  16. Labeling and preliminary in vivo assessment of niobium-labeled radioactive species: A proof-of-concept study.

    PubMed

    Radchenko, Valery; Bouziotis, Penelope; Tsotakos, Theodoros; Paravatou-Petsotas, Mari; la Fuente, Ana de; Loudos, George; Harris, Adrian L; Xanthopoulos, Stavros; Filosofov, Dmitry; Hauser, Harald; Eisenhut, Michael; Ponsard, Bernard; Roesch, Frank

    2016-05-01

    The application of radionuclide-labeled biomolecules such as monoclonal antibodies or antibody fragments for imaging purposes is called immunoscintigraphy. More specifically, when the nuclides used are positron emitters, such as zirconium-89, the technique is referred to as immuno-PET. Currently, there is an urgent need for radionuclides with a half-life which correlates well with the biological kinetics of the biomolecules under question and which can be attached to the proteins by robust labeling chemistry. (90)Nb is a promising candidate for in vivo immuno-PET, due its half-life of 14.6h and low β(+) energy of Emean=0.35MeV per decay. (95)Nb on the other hand, is a convenient alternative for longer-term ex vivo biodistribution studies, due to its longer half-life of (t½=35days) and its convenient, lower-cost production (reactor-based production). In this proof-of-principle work, the monoclonal antibody bevacizumab (Avastin(®)) was labeled with (95/90)Nb and in vitro and in vivo stability was evaluated in normal Swiss mice and in tumor-bearing SCID mice. Initial ex vivo experiments with (95)Nb-bevacizumab showed adequate tumor uptake, however at the same time high uptake in the liver, spleen and kidneys was observed. In order to investigate whether this behavior is due to instability of (⁎)Nb-bevacizumab or to the creation of other (⁎)Nb species in vivo, we performed biodistribution studies of (95)Nb-oxalate, (95)Nb-chloride and (95)Nb-Df. These potential metabolite species did not show any specific uptake, apart from bone accumulation for (95)Nb-oxalate and (95)Nb-chloride, which, interestingly, may serve as an "indicator" for the release of (90)Nb from labeled biomolecules. Concerning the initial uptake of (95)Nb-bevacizumab in non-tumor tissue, biodistribution of a higher specific activity radiolabeled antibody sample did show only negligible uptake in the liver, spleen, kidneys or bones. In-vivo imaging of a tumor-bearing SCID mouse after injection

  17. Do Low 15N Values in Paleozoic Epeiric Basins Indicate High Rates of N Fixation?

    NASA Astrophysics Data System (ADS)

    Tuite, M. L.; Macko, S. A.

    2011-12-01

    As a consequence of the high energetic requirements of dinitrogen (N2) fixation, organic N produced by diazotrophic microorganisms typically exhibits δ15N values similar to atmospheric N2 (approximately 0%). Because the δ15N of organic-rich Paleozoic epeiric basin sediments often has values in the vicinity of 0%, it is frequently asserted that N2 fixation was the primary source of new reactive N for productivity. There are two broad reasons why recourse to widespread and intensive N fixation as the primary source of the organic N is problematic. First, there are substantial physiological and ecological constraints on marine N fixation that limit its extent in modern oceans primarily to open ocean basins. Second, preservation of an unaltered isotopic signature of diazotrophy in underlying sediments is not a likely outcome of oxic and anoxic diagenetic alteration and repeated cycles of mineralization and assimilation. Constraining the sources of reactive N for primary productivity is critical to understanding the N cycle in Paleozoic epeiric seas. In this study we report δ15N values from high organic matter Middle Ordovician through Late Devonian dysoxic and euxinic basinal sediments. We propose a nitrogen isotope mass balance model that incorporates the microbial ecology of a stratified water column and the biochemical stoichiometry of primary production and organic matter diagenesis. Results from the model support our contention that high rates of N fixation over extended time periods were not the cause of depleted nitrogen isotope values in organic-rich Paleozoic basinal sediments. Rather, the depleted values were a consequence of a diminished role for nitrification and subsequent N loss via denitrification and anammox, and the preferential preservation of a substantially 15N-depleted chlorophyll-influenced lipid fraction. The model may be applicable to earlier and later geological periods where high organic matter sediments feature depleted δ15N values.

  18. [Characteristics of urea 15N absorption, allocation, and utilization by sweet-cherry (Prunus avium L.)].

    PubMed

    Zhao, Feng-Xia; Jiang, Yuan-Mao; Peng, Fu-Tian; Gao, Xiang-Bin; Liu, Bing-Hua; Wang, Hai-Yun; Zhao, Lin

    2008-03-01

    With five-year old 'Zaodaguo' sweet-cherry (Prunus avium L.) as test material, this paper studied the characteristics of its urea 15N absorption, allocation, and utilization when applied before bud-break. The results showed that the Ndff of different organs increased gradually with time, and was higher in fine roots and storage organs at full-blooming stage. At fruit core-hardening stage, the Ndff of long shoots and leaves increased quickly, reaching to 0.72% and 0.59% , respectively. From fruit core-hardening to harvesting stage, the Ndff of fruit had a rapid increase, with the peak (1.78%) at harvesting stage. After harvest, the Ndff of neonatal organs increased slowly while that of storage organs increased quickly. At full-blooming stage, the absorbed 15N in roots was firstly allocated to storage organs, with the highest allocation rate (54.91%) in large roots. At fruit core-hardening stage, the allocation rate in fine roots and storage organs decreased from 85.43% to 55.11%, while that in neonatal branches and leaves increased to 44.89%. At harvesting stage, the allocation rate in different organs had no significant change, but after harvest, the absorbed 15N had a rapid translocation to storage organs, and the allocation rate in fine roots and storage organs reached the highest (72.26%) at flower bud differentiation stage. The 15N allocation rate in neonatal branches and leaves at flower bud differentiation stage was decreased by 19.31%, compared with that at harvesting stage. From full-blooming to flower bud differentiation stage, the utilization rate of urea 15N was increasing, and reached the peak (16.86%) at flower bud differentiation stage.

  19. Tocotrienol Treatment in Familial Dysautonomia: Open-Label Pilot Study.

    PubMed

    Cheishvili, David; Maayan, Channa; Holzer, Naama; Tsenter, Jeanna; Lax, Elad; Petropoulos, Sophie; Razin, Aharon

    2016-07-01

    Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy, primarily presented in Ashkenazi Jews. The most common mutation in FD patients results from a single base pair substitution of an intronic splice site in the IKBKAP gene which disrupts normal mRNA s