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Sample records for 15n metabolic labeling

  1. Automated Protein Turnover Calculations from 15N Partial Metabolic Labeling LC/MS Shotgun Proteomics Data

    PubMed Central

    Lyon, David; Castillejo, Maria Angeles; Staudinger, Christiana; Weckwerth, Wolfram; Wienkoop, Stefanie; Egelhofer, Volker

    2014-01-01

    Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent protein turnover calculations enable the analysis of gigabytes of data within minutes, a prerequisite for systems biology high throughput studies. Here we present a fully automated method for protein turnover calculations from shotgun proteomics data. The approach enables the analysis of complex shotgun LC/MS 15N partial metabolic labeling experiments. Spectral envelopes of 1419 peptides can be extracted within an hour. The method quantifies turnover by calculating the Relative Isotope Abundance (RIA), which is defined as the ratio between the intensity sum of all heavy (15N) to the intensity sum of all light (14N) and heavy peaks. To facilitate this process, we have developed a computer program based on our method, which is freely available to download at http://promex.pph.univie.ac.at/protover. PMID:24736476

  2. A pipeline for 15N metabolic labeling and phosphoproteome analysis in Arabidopsis thaliana.

    PubMed

    Minkoff, Benjamin B; Burch, Heather L; Sussman, Michael R

    2014-01-01

    Within the past two decades, the biological application of mass spectrometric technology has seen great advances in terms of innovations in hardware, software, and reagents. Concurrently, the burgeoning field of proteomics has followed closely (Yates et al., Annu Rev Biomed Eng 11:49-79, 2009)-and with it, importantly, the ability to globally assay altered levels of posttranslational modifications in response to a variety of stimuli. Though many posttranslational modifications have been described, a major focus of these efforts has been protein-level phosphorylation of serine, threonine, and tyrosine residues (Schreiber et al., Proteomics 8:4416-4432, 2008). The desire to examine changes across signal transduction cascades and networks in their entirety using a single mass spectrometric analysis accounts for this push-namely, preservation and enrichment of the transient yet informative phosphoryl side group. Analyzing global changes in phosphorylation allows inferences surrounding cascades/networks as a whole to be made. Towards this same end, much work has explored ways to permit quantitation and combine experimental samples such that more than one replicate or experimental condition can be identically processed and analyzed, cutting down on experimental and instrument variability, in addition to instrument run time. One such technique that has emerged is metabolic labeling (Gouw et al., Mol Cell Proteomics 9:11-24, 2010), wherein biological samples are labeled in living cells with nonradioactive heavy isotopes such as (15)N or (13)C. Since metabolic labeling in living organisms allows one to combine the material to be processed at the earliest possible step, before the tissue is homogenized, it provides a unique and excellent method for comparing experimental samples in a high-throughput, reproducible fashion with minimal technical variability. This chapter describes a pipeline used for labeling living Arabidopsis thaliana plants with nitrogen-15 ((15)N) and how

  3. 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

  4. A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry.

    PubMed

    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 ((14)N/(15)N) 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

  5. Rapid and automated processing of MALDI-FTICR/MS data for 15N-metabolic labeling in a shotgun proteomics analysis

    NASA Astrophysics Data System (ADS)

    Jing, Li; Amster, I. Jonathan

    2009-10-01

    Offline high performance liquid chromatography combined with matrix assisted laser desorption and Fourier transform ion cyclotron resonance mass spectrometry (HPLC-MALDI-FTICR/MS) provides the means to rapidly analyze complex mixtures of peptides, such as those produced by proteolytic digestion of a proteome. This method is particularly useful for making quantitative measurements of changes in protein expression by using 15N-metabolic labeling. Proteolytic digestion of combined labeled and unlabeled proteomes produces complex mixtures with many mass overlaps when analyzed by HPLC-MALDI-FTICR/MS. A significant challenge to data analysis is the matching of pairs of peaks which represent an unlabeled peptide and its labeled counterpart. We have developed an algorithm and incorporated it into a computer program which significantly accelerates the interpretation of 15N-metabolic labeling data by automating the process of identifying unlabeled/labeled peak pairs. The algorithm takes advantage of the high resolution and mass accuracy of FTICR mass spectrometry. The algorithm is shown to be able to successfully identify the 15N/14N peptide pairs and calculate peptide relative abundance ratios in highly complex mixtures from the proteolytic digest of a whole organism protein extract.

  6. 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

  7. 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. PMID:22543713

  8. Heavy water and (15) N labelling with NanoSIMS analysis reveals growth rate-dependent metabolic heterogeneity in chemostats.

    PubMed

    Kopf, Sebastian H; McGlynn, Shawn E; Green-Saxena, Abigail; Guan, Yunbin; Newman, Dianne K; Orphan, Victoria J

    2015-07-01

    To measure single-cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and (15) N 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 % (2) H). 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 h, 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 versus 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

  9. 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

  10. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Studies with 15N-labeled ammonia and urea in the malnourished child

    PubMed Central

    Read, W. W. C.; McLaren, D. S.; Tchalian, Marie; Nassar, Siham

    1969-01-01

    Investigations using ammonium citrate-15N and urea-15N showed that children in the acute stage of kwashiorkor and marasmus receiving a diet of adequate protein content retained a considerable percentage of the label from both compounds. Excretion of both total 15N and urea-15N was subnormal and elimination was virtually completed 36 hr after administration of the isotope. During recovery from kwashiorkor total 15N excretion had approached normal a month after commencement of rehabilitation. Urea-15N excretion was still slightly subnormal after 3 months. In marasmus urea-15N formed a normal proportion of total 15N excretion after 1 month, although total 15N excretion then was still low. Ammonia nitrogen was retained to a greater extent than urea nitrogen in all cases. As it is known that a considerable amount of urea is degraded to ammonia in the gastrointestinal tract, it seems probable that urea nitrogen became available for use after this degradation. Examination of blood from one marasmic child after feeding ammonia-15N and from another after intravenous injection of urea-15N showed incorporation of the label into blood cells and plasma proteins. This did not occur in well nourished controls. It is concluded that ammonia and urea as sources of nonessential nitrogen may play an important part in protein metabolism in the malnourished child. PMID:5771193

  13. 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

  14. 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. PMID:24359440

  15. Isolation and measurement of 15N2 from respiratory gases of animals administered 15N-labeled substances.

    PubMed

    Springer, D L; Reed, D J; Dost, F N

    1981-01-01

    A method is described for collection of metabolic 15N2 from in vitro preparations or intact rats administered 15N-containing compounds. The methods enables routine collection and mass spectrometric measurement of as little as 10 mumol 15N2 respired by a rat over a 24-h period. A device is described that includes either an animal chamber or a tissue reaction vessel in a closed recycling atmosphere, with automatic O2 replenishment and removal of CO2 and water. It is capable of sustaining moderate vacuum and is coupled to a high-vacuum manifold designed to process the contained atmosphere and respiratory gases. The starting atmosphere is an 80:20 mix of sulfur hexafluoride and O2. Recovery of 15N2 gas from the system without an animal present was 101.3 +/- 5.75%. When 15N2 gas was very slowly infused iv into an animal, recovery was 89.1 +/- 5.38%. Use of the method in studies of the fate of [15N]hydrazine in rats indicated that about 15% of the administered hydrazine is rapidly converted to 15N2, followed by slower conversion of an additional 7-10% over the next several hours. PMID:7328697

  16. Biosynthetic uniform 13C,15N-labelling of zervamicin IIB. Complete 13C and 15N NMR assignment.

    PubMed

    Ovchinnikova, Tatyana V; Shenkarev, Zakhar O; Yakimenko, Zoya A; Svishcheva, Natalia V; Tagaev, Andrey A; Skladnev, Dmitry A; Arseniev, Alexander S

    2003-01-01

    Zervamicin IIB is a member of the alpha-aminoisobutyric acid containing peptaibol antibiotics. A new procedure for the biosynthetic preparation of the uniformly 13C- and 15N-enriched peptaibol is described This compound was isolated from the biomass of the fungus-producer Emericellopsis salmosynnemata strain 336 IMI 58330 obtained upon cultivation in the totally 13C, 15N-labelled complete medium. To prepare such a medium the autolysed biomass and the exopolysaccharides of the obligate methylotrophic bacterium Methylobacillus flagellatus KT were used. This microorganism was grown in totally 13C, 15N-labelled minimal medium containing 13C-methanol and 15N-ammonium chloride as the only carbon and nitrogen sources. Preliminary NMR spectroscopic analysis indicated a high extent of isotope incorporation (> 90%) and led to the complete 13C- and 15N-NMR assignment including the stereospecific assignment of Aib residues methyl groups. The observed pattern of the structurally important secondary chemical shifts of 1H(alpha), 13C=O and 13C(alpha) agrees well with the previously determined structure of zervamicin IIB in methanol solution. PMID:14658801

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. 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.

  19. Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins.

    PubMed

    Pintacuda, Guido; Keniry, Max A; Huber, Thomas; Park, Ah Young; Dixon, Nicholas E; Otting, Gottfried

    2004-03-10

    A novel strategy for fast NMR resonance assignment of (15)N HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively (15)N-labeled samples. Comparison of sensitive undecoupled (15)N HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia coli DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy. PMID:14995214

  20. The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements.

    PubMed

    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

  1. Whole body nitric oxide synthesis in healthy men determined from [15N] arginine-to-[15N]citrulline labeling.

    PubMed Central

    Castillo, L; Beaumier, L; Ajami, A M; Young, V R

    1996-01-01

    The rates of whole body nitric oxide (NO) synthesis, plasma arginine flux, and de novo arginine synthesis and their relationships to urea production, were examined in a total of seven healthy adults receiving an L-amino acid diet for 6 days. NO synthesis was estimated by the rate of conversion of the [15N] guanidino nitrogen of arginine to plasma [15N] ureido citrulline and compared with that based on urinary nitrite (NO2-)/nitrate (NO3-) excretion. Six subjects received on dietary day 7, a 24-hr (12-hr fed/12-hr fasted) primed, constant, intravenous infusion of L-[guanidino-15N2]arginine and [13C]urea. A similar investigation was repeated with three of these subjects, plus an additional subject, in which they received L-[ureido-13C]citrulline, to determine plasma citrulline fluxes. The estimated rates (mean +/- SD) of NO synthesis over a period of 24 hr averaged 0.96 +/- 0.1 mumol .kg-1.hr-1 and 0.95 +/- 0.1 mumol.kg-1.hr-1, for the [15N]citrulline and the nitrite/nitrate methods, respectively. About 15% of the plasma arginine turnover was associated with urea formation and 1.2% with NO formation. De novo arginine synthesis averaged 9.2 +/- 1.4 mumol. kg-1.hr-1, indicating that approximately 11% of the plasma arginine flux originates via conversion of plasma citrulline to arginine. Thus, the fraction of the plasma arginine flux associated with NO and also urea synthesis in healthy humans is small, although the plasma arginine compartment serves as a significant precursor pool (54%) for whole body NO formation. This tracer model should be useful for exploring these metabolic relationships in vivo, under specific pathophysiologic states where the L-arginine-NO pathway might be altered. Images Fig. 4 PMID:8876157

  2. 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.

  3. 15N labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons

    PubMed Central

    Liao, Lujian; Sando, Richard C.; Farnum, John B.; Vanderklish, Peter W.; Maximov, Anton; Yates, John R.

    2011-01-01

    Terminally differentiated primary cells represent a valuable in vitro model to study signaling events associated within a specific tissue. Quantitative proteomic methods using metabolic labeling in primary cells encounter labeling efficiency issues hindering the use of these cells. Here we developed a method to quantify the proteome and phosphoproteome of cultured neurons using 15N labeled brain tissue as an internal standard, and applied this method to determine how an inhibitor of an excitatory neural transmitter receptor, phencyclidine (PCP), affects the global phosphoproteome of cortical neurons. We identified over 10,000 phosphopeptides and made accurate quantitative measurements of the neuronal phosphoproteome after neuronal inhibition. We show that short PCP treatments lead to changes in phosphorylation for 7% of neuronal phosphopeptides and that prolonged PCP treatment alters the total levels of several proteins essential for synaptic transmission and plasticity and leads to a massive reduction in the synaptic strength of inhibitory synapses. The results provide valuable insights into the dynamics of molecular networks implicated in PCP-mediated NMDA receptor inhibition and sensorimotor deficits. PMID:22070516

  4. Numerical evaluation of subsoil diffusion of (15) N labelled denitrification products during employment of the (15) N gas flux method in the field

    NASA Astrophysics Data System (ADS)

    Well, Reinhard; Buchen, Caroline; Lewicka-Szczebak, Dominika; Ruoss, Nicolas

    2016-04-01

    Common methods for measuring soil denitrification in situ include monitoring the accumulation of 15N labelled N2 and N2O evolved from 15N labelled soil nitrate pool in soil surface chambers. Gas diffusion is considered to be the main accumulation process. Because accumulation of the gases decreases concentration gradients between soil and chamber over time, gas production rates are underestimated if calculated from chamber concentrations. Moreover, concentration gradients to the non-labelled subsoil exist, inevitably causing downward diffusion of 15N labelled denitrification products. A numerical model for simulating gas diffusion in soil was used in order to determine the significance of this source of error. Results show that subsoil diffusion of 15N labelled N2 and N2O - and thus potential underestimation of denitrification derived from chamber fluxes - increases with cover closure time as well as with increasing diffusivity. Simulations based on the range of typical gas diffusivities of unsaturated soils show that the fraction of subsoil diffusion after chamber closure for 1 hour is always significant with values up to >30 % of total production of 15N labelled N2 and N2O. Field experiments for measuring denitrification with the 15N gas flux method were conducted. The ability of the model to predict the time pattern of gas accumulation was evaluated by comparing measured 15N2 concentrations and simulated values.

  5. Spectroscopic labeling of A, S/T in the 1H- 15N HSQC spectrum of uniformly ( 15N- 13C) labeled proteins

    NASA Astrophysics Data System (ADS)

    Chugh, Jeetender; Hosur, Ramakrishna V.

    2008-10-01

    A new triple resonance two-dimensional experiment, termed (HC)NH, has been described to generate specific labels on the peaks of alanines and serines/threonines, separately, in the 1H- 15N HSQC spectrum of a protein. The performance of the pulse sequence has been demonstrated with a 151 residue protein. The method permits the investigation of local environments around those specific residues without actually having to obtain complete resonance assignments for the entire protein. With this one can envisage use of the technique for studying large protein systems from different points of view.

  6. Fate and metabolism of [15N]2,4,6-trinitrotoluene in soil.

    PubMed

    Weiss, Martin; Geyer, Roland; Russow, Rolf; Richnow, Hans H; Kästner, Matthias

    2004-08-01

    The fates of the labels from [14C] and [15N] trinitrotoluene were analyzed in bioreactors under aerobic conditions in soil treated by a fungal bioremediation process with Stropharia rugosoannulata and in control soil. Up to 17.5% of the 15N label had a different fate than the 14C label. Three N-mineralization processes were identified in detailed experiments with [15N]TNT. About 2% of the 15N label was found as NO3- and NH4+, showing simultaneous processes of direct TNT denitration (I) and reduction with cleavage of the amino groups (II). The enrichment of NO2-/NO3- (up to 7.5 atom% 15N abundance) indicates the formation of Meisenheimer complexes with a denitration of [15N]TNT. A 1.4% of the label was found distributed between N2O and N2. However, the 15N enrichment of the N2O (up to 38 atom%) demonstrated that both N atoms were generated from the labeled TNT and clearly indicates a novel formation process (III). We propose, as an explanation, the generation of N2O by cleavage from condensed azoxy metabolites. In addition, 1.7% of the 15N label was detected as biogenic amino acids in the wheat straw containing the fungus. Overall, 60 to 85% of the applied [15N]TNT was degraded and 52 to 64% was found as nonextractable residues in the soil matrix. Three percent was detected as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene. PMID:15352472

  7. Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies.

    PubMed

    Nikonowicz, E P; Sirr, A; Legault, P; Jucker, F M; Baer, L M; Pardi, A

    1992-09-11

    A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence. Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media. These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose. The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription. Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC). Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively. These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues. The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs. Examples of several such heteronuclear NMR experiments are shown. PMID:1383927

  8. Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized 1H and 15N NMR spectroscopy

    PubMed Central

    Cudalbu, Cristina; Lanz, Bernard; Duarte, João MN; Morgenthaler, Florence D; Pilloud, Yves; Mlynárik, Vladimir; Gruetter, Rolf

    2012-01-01

    Brain glutamine synthetase (GS) is an integral part of the glutamate–glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. 15N MRS is an alternative approach to 13C MRS. Incorporation of labeled 15N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate–glutamine cycle. To measure 15N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel 15N pulse sequence to simultaneously detect, for the first time, [5-15N]Gln and [2-15N]Gln+Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized 1H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of 1H and 15N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30±0.050 μmol/g per minute), apparent neurotransmission (0.26±0.030 μmol/g per minute), glutamate dehydrogenase (0.029±0.002 μmol/g per minute), and net glutamine accumulation (0.033±0.001 μmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification. PMID:22167234

  9. Isolation and measurement of /sup 15/N/sub 2/ from respiratory gases of animals administered /sup 15/N-labeled substances

    SciTech Connect

    Springer, D.L.; Reed, D.J.; Dost, F.N.

    1981-07-01

    A method is described for collection of metabolic /sup 15/N/sub 2/ from in vitro preparations or intact rats administered /sup 15/N-containing compounds. The method enables routine collection and mass spectrometric measurement of as little as 10 ..mu..mol /sup 15/N/sub 2/ respired by a rat over a 24-h period. A device is described that includes either an animal chamber or a tissue reaction vessel in a closed recycling atmosphere, with automatic O/sub 2/ replenishment and removal of CO/sub 2/ and water. It is capable of sustaining moderate vacuum and is coupled to a high-vacuum manifold designed to process the contained atmosphere and respiratory gases. The starting atmosphere is an 80:20 mix of sulfur hexafluoride and O/sub 2/. Recovery of /sup 15/N/sub 2/ gas from the system without an animal present was 101.3 +/- 5.75%. When /sup 15/N/sub 2/ gas was very slowly infused iv into an animal, recovery was 89.1 +/- 5.38%. Use of the method in studies of the fate of (/sup 15/N)hydrazine in rats indicated that about 15% of the administered hydrazine is rapidly converted to /sup 15/N/sub 2/, followed by slower conversion of an additional 7-10% over the next several hours.

  10. 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.

  11. HN-NCA heteronuclear TOCSY-NH experiment for (1)H(N) and (15)N sequential correlations in ((13)C, (15)N) labelled intrinsically disordered proteins.

    PubMed

    Wiedemann, Christoph; Goradia, Nishit; Häfner, Sabine; Herbst, Christian; Görlach, Matthias; Ohlenschläger, Oliver; Ramachandran, Ramadurai

    2015-10-01

    A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue 'i' with that of residues 'i-1' and 'i+1' in ((13)C, (15)N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of (1) J CαN and (2) J CαN couplings to transfer the (15)N x magnetisation from amino acid residue 'i' to adjacent residues via the application of a band-selective (15)N-(13)C(α) heteronuclear cross-polarisation sequence of ~100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described. PMID:26282620

  12. Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring.

    PubMed

    Bouhss, A; Sakamoto, H; Palibroda, N; Chiriac, M; Sarfati, R; Smith, J M; Craescu, C T; Bârzu, O

    1995-02-10

    GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N. PMID:7778777

  13. 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

  14. 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+).

  15. 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

  16. Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium.

    PubMed

    Englander, Jacqueline; Cohen, Leah; Arshava, Boris; Estephan, Racha; Becker, Jeffrey M; Naider, Fred

    2006-01-01

    Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids. Growth in minimal media often results in low yields of cells or expression products. We demonstrate a method in which one labeled amino acid is added to a rich medium. These conditions resulted in high expression (> or =100 mg/L) of a test fusion protein and milligram quantities of the selectively labeled membrane peptide after cyanogen bromide cleavage to release the peptide from the fusion protein. High levels of (15)N incorporation and acceptable levels of cross-labeling into other amino acid residues of the peptide were achieved. Growth in rich media is a simple and convenient alternative to growth in supplemented minimal media and is readily applicable to the expression of proteins selectively labeled with specific amino acids. PMID:16741986

  17. Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

    PubMed Central

    Masse, J E; Bortmann, P; Dieckmann, T; Feigon, J

    1998-01-01

    The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures. PMID:9592146

  18. 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.

  19. Exogenous proline relieves growth inhibition caused by NaCl in petunia cells: Metabolism of L-( sup 15 M)-proline followed by sup 15 N NMR

    SciTech Connect

    Heyser, J.W.; Chacon, M.J. )

    1989-04-01

    Exogenous proline stimulated the growth of Petunia hybrida cells on 195 mM NaCl 10-fold as compared with cells grown on 195 mM CaCl medium minus proline. L-({sup 15}N)-proline was fed to cells growing on 0 and 195 mM CaCl, and its metabolism was followed by {sup 15}N NMR analysis of cell extracts. Total proline and amino acids were determined by ninhydrin assay. Proline and primary amino acids were easily resolved in NMR spectra and the amount of {sup 15}N-label which remained in proline was determined. Reduced catabolism of proline in cells grown on NaCl was evident. The role of exogenous proline in conferring increased NaCl tolerance in this nonhalophyte will be discussed.

  20. 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.

  1. High resolution (13)C MRI with hyperpolarized urea: in vivo T(2) mapping and (15)N labeling effects.

    PubMed

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

    2014-02-01

    (13)C steady state free precession (SSFP) magnetic resonance imaging and effective spin-spin relaxation time (T2) mapping were performed using hyperpolarized [(13)C] urea and [(13) C,(15)N2] urea injected intravenously in rats. (15)N 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 [(13) C,(15) N2] 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. [(13)C] 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 [(13)C,(15)N2] 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 [(13)C,(15) N2] urea giving a greater than four-fold increase in signal-to-noise ratio over [(13)C] urea. PMID:24235273

  2. /sup 15/N and /sup 13/C NMR determination of methionine metabolism in developing soybean cotyledons

    SciTech Connect

    Coker, G.T. III; Garbow, J.R.; Schaefer, J.

    1987-03-01

    The metabolism of D- and L-methionine by immature cotyledons of soybean (Glycine max, L. cv Elf) grown in culture has been investigated using solid-state /sup 13/C and /sup 15/N nuclear magnetic resonance. D-Methionine is taken up by the cotyledons and converted to an amide, most likely by N-malonylation. About 16% of the L-methionine taken up is incorporated intact into protein, and 25% remains as soluble methionine. Almost two-thirds of the L-methionine that enters the cotyledons is degraded. The largest percentage of this is used in transmethylation of the carboxyl groups of pectin. Methionine is not extensively converted to polyamines. The authors attribute the stimulation of growth of the cotyledons by exogenous methionine to the bypassing of a rate-limiting methyl-transfer step in the synthesis of methionine itself, and subsequently of pectins and proteins.

  3. 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

  4. 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. PMID:26982183

  5. 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

  6. 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

  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

    Quantifying dinitrogen (N2) and nitrous oxide (N2O) fluxes from different soil N pools and processes can be accomplished using the 15N tracer technique but this is subject to four different sources of bias (i. - iv.). This approach includes 15N labelling of selected N pools in soil and subsequent isotope analysis of all relevant N pools as well as of gas samples from enclosures, i.e. mixtures of soil-derived and atmospheric N2 and N2O. Depending on the processes of interest, there may be 15N labelling of one or several N pools, were several labelling treatment are needed in the latter case (e.g. Müller et al., 2004). Measuring pool-derived N2 or N2O has been shown to include two calculation problems, (i.) arising from multiple pools (e.g. Arah, 1992) and (ii.) dealing with the non-random distribution of N2 and N2O mole masses (Hauck et al., 1958). Non-randomness can be solved if m/z 28, 29 and 30 are correctly analysed and the 15N enrichment of one (to distinguish two pools, i.e. soil and atmosphere) or two pools (in case of three pools) is known (Spott & Stange, 2008). Moreover (iii.), NO3- pools generating N2 and N2O via denitrification can be identical or different, e.g. if N2O evolved from higher enriched NO3- in deeper soil was more reduced to N2 compared to N2O evolved from N2O from shallow soil with lower enrichment, or vice versa. Apportioning N2O fluxes to NH4+ (nitrification and/or nitrifier denitrification) and NO3- (denitrification) is often conducted by NO3-labeling, measuring δ15N of emitted N2O and applying mixing equations were the measured 15N enrichment of NH4+and NO3-pool is used. However, this assumes that the average 15N enrichment of NH4+and NO3-in the soil is identical to the enrichment in the active soil domain producing N2 and/or N2O. Violation of this precondition must lead to bias in source apportionment (iv.), but to our knowledge this has not been investigated until now. Here we present conceptual models and model calculations

  8. Quantitation of metabolic compartmentation in hyperammonemic brain by natural abundance 13C-NMR detection of 13C-15N coupling patterns and isotopic shifts.

    PubMed

    Lapidot, A; Gopher, A

    1997-02-01

    In the present study, the removal of cerebral ammonia by glutamine synthetase (GS) and by reductive amination of 2-oxoglutarate by glutamate dehydrogenase in the presence of an amino donor group, was determined in hyperammonemic rabbit brains. The 15N enrichments of brain metabolite alpha-amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of 15N-labeled compounds of the 13C-15N spin coupling patterns of natural abundance 13C-NMR spectra. The 13C-NMR spectra of brain extracts were obtained from rabbits infused with 15NH4Cl with or without intraperitoneal infusion of the GS inhibitor, L-methionine DL-sulfoximine, in a reasonable acquisition time period. When 15NH4Cl was infused, [5-15N]glutamine and [2-15N]glutamine concentrations reached 5.2 mumol/100 mg protein and 3.6 mumol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. The low concentration of [2-15N]glutamate, which is about 30% of that of [2-15N]glutamine obtained in this study, suggests that very little glutamine serves as a precursor of neuronal glutamate. When GS was inhibited by L-methionine DL-sulfoximine, a flux of 15NH4+ via the residual activity of GS was accompanied by an apparent increase of [2-15N]glutamate and [15N]alanine concentrations (2.9 mumol/100 mg protein and 1.8 mumol/100 mg protein, respectively). These findings and those obtained from 13C-13C isotopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2-oxoglutarate is partially utilized (together with an amino group donor) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in metabolizing ammonia in the hyperammonemic rabbit brain. PMID:9057821

  9. Identification of the magnesium-histidine stretching vibration of the bacteriochlorophyll cofactors in photosynthetic reaction centers via {sup 15}N-labeling of the histidines

    SciTech Connect

    Czarnecki, K.; Bocian, D.F.; Chynwat, V.; Erickson, J.P.; Frank, H.A.

    1997-03-12

    In this communication, we report low-frequency, near-infrared-exciation RR spectra of bacterial RCs in which the histidine residues of the protein are selectively labeled with {sup 15}N. For practical reasons, the studies were conducted by comparing the vibrational signatures of RCs in which {sup 15}N was universally incorporated (all cofactors and all protein residues) (designated all-{sup 15}N RCs) with those in which [{sup 14}N]histidine was introduced as a reverse label (disignated {sup 14}N-His RCs) into the all {sup 15}N-labeled RCs. The studies of the histidine-labeled RCs reveal that the vibrational characteristics of the BChl core are far more complicated than originally anticipated. These results have clear implications for the photoexcitation dynamics of the BChls in RCs and may also have significant consequences for the dynamics of exogenous ligand binding to heme-based oxygen carriers. 12 refs., 2 figs.

  10. 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

  11. Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements

    PubMed Central

    Jaruga, Pawel; Nelson, Bryant C.; Lowenthal, Mark S.; Jemth, Ann-Sofie; Loseva, Olga; Coskun, Erdem; Helleday, Thomas

    2016-01-01

    Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA–protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length 15N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of 15N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented. PMID:26791985

  12. 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.

  13. 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. PMID:25973921

  14. 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

  15. Incorporation of 15N-labeled ammonia into glutamine amide groups by protein-glutaminase and analysis of the reactivity for α-lactalbumin.

    PubMed

    Miwa, Noriko; Shimba, Nobuhisa; Nakamura, Mina; Yokoyama, Keiichi; Nio, Noriki; Suzuki, Eiichiro; Sonomoto, Kenji

    2011-12-28

    Protein-glutaminase (PG) is an enzyme that catalyzes the deamidation of protein-bound glutamine residues. We found that an enzyme labeling technique (ELT), which is a stable isotope labeling method based on transglutaminase (TGase) reaction, is applicable for PG. PG catalyzed incorporation of (15)N-labeled ammonium ions into reactive glutamine amide groups in α-lactalbumin similarly to TGase and deamidated the most reactive glutamine amide group once labeled with (15)N. Furthermore, we investigated the effect of ammonium ions on the PG activity by peptide mapping, and more reactive glutamine residues were detected than were detected by the ELT in the presence of ammonium ions. This is probably because ammonium ions are competitive inhibitors, causing decreased reactivity for glutamine residues. We propose the reaction scheme of PG in the presence of the (15)N-labeled ammonium ions and show that the ELT method with PG is useful for evaluating the activity of PG. PMID:22060122

  16. Mapping membrane protein backbone dynamics: a comparison of site-directed spin labeling with NMR 15N-relaxation measurements.

    PubMed

    Lo, Ryan H; Kroncke, Brett M; Solomon, Tsega L; Columbus, Linda

    2014-10-01

    The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins. PMID:25296323

  17. Evidence for tautomerism in nucleic acid base pairs. 1H NMR study of 15N labeled tRNA.

    PubMed Central

    Rüterjans, H; Kaun, E; Hull, W E; Limbach, H H

    1982-01-01

    The imino proton resonances of 15N labeled tRNA appear as asymmetric doublet signals, the asymmetry being dependent on the applied magnetic field strength. Assuming a tautomerism of the type N-H...N not equal to N...H-N in the base pairs the line shapes can be simulated. The most important parameters fitted in the simulation are the rate constants of the proton transfer and the mole fractions of either tautomeric state. The rate constants are of the order of 100s-1 and the mole fractions of the non dominant tautomer about 0.1 depending on the temperature and on the nature of the base pairing. The observations are attributed to a double proton transfer in the base pairs. The unexpectedly slow rates of the double proton transfer process may be connected with a concomitant conformational change of the duplex structure. PMID:7177856

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

    PubMed

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

    2015-12-01

    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 (15)N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N 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 (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N 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 (15)N and (13)C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants. PMID:26231074

  19. An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts.

    PubMed

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-07-01

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein (15)N and (13)C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor. PMID:26070442

  20. 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.

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

    PubMed

    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 (13)C,(15)N-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, 22kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600MHz. 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. PMID:20060344

  2. 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. PMID:24186940

  3. 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.

  4. 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

  5. High Field Solid-State NMR Spectroscopy Investigation of (15)N-Labeled Rosette Nanotubes: Hydrogen Bond Network and Channel-Bound Water.

    PubMed

    Fenniri, Hicham; Tikhomirov, Grigory A; Brouwer, Darren H; Bouatra, Souhaila; El Bakkari, Mounir; Yan, Zhimin; Cho, Jae-Young; Yamazaki, Takeshi

    2016-05-18

    (15)N-labeled rosette nanotubes were synthesized and investigated using high-field solid-state NMR spectroscopy, X-ray diffraction, atomic force microscopy, and electron microscopy. The results established the H-bond network involved in the self-assembly of the nanostructure as well as bound water molecules in the nanotube's channel. PMID:27141817

  6. ESR characterization of a novel spin-trapping agent, 15N-labeled N-tert-butyl-alpha-phenylnitrone, as a nitric oxide donor.

    PubMed

    Saito, Kieko; Yoshioka, Hisashi

    2002-10-01

    We previously found that one of the pharmacological effects of N-tert-butyl-alpha-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled 15N to identify its corresponding 15NO in vivo. The properties were examined with an ESR spectrometer. To synthesize 15N-PBN, the starting material, ammonium-15N chloride, was converted to 2-amino-15N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-15N, and finally reacted with benzaldehyde to give 15N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)2, as a trapping agent to measure the NO levels of 15N-PBN or 14N-PBN in vitro, the peak intensity of 15NO[Fe(MGD)2] was over 50% stronger than that of 14NO[Fe(MGD)2], and that 15NO and 14NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of 15NO derived from 15N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish 15NO from PBN and 14NO generated from cells. These results suggested that 15N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo. PMID:12450131

  7. Selective observation of biologically important 15N-labeled metabolites in isolated rat brain and liver by 1H-detected multiple-quantum-coherence spectroscopy

    NASA Astrophysics Data System (ADS)

    Kanamori, Keiko; Ross, Brian D.; Parivar, Farhad

    Four cerebral metabolites of importance in neurotransmission, serotonin, L-tryptophan, L-glutamine, and N-acetyl- L-aspartate, and two hepatic urea-cycle intermediates, citrulline and urea, were found to be observable by 1H- 15N heteronuclear multiple-quantum-coherence (HMQC) spectroscopy in aqueous solution at physiological pH and temperature, through the protons spin-coupled to their indole, amide, or ureido nitrogen. Their 1H chemical shifts were well dispersed over a 5-10 ppm region while the 1J 15N- 1H values were 87-99 Hz. For [γ- 15N]glutamine, a 50- to 100-fold increase in sensitivity over direct 15N detection was achieved, in contrast to a 2-fold increase by the polarization-transfer method. In the isolated brain of portacaval-shunted rats, the amide protons of biologically 15N-enriched [γ- 15N]glutamine were observed in 2 min of acquisition, with suppression of proton signals from all other cerebral metabolites. In isolated liver of 15N-enriched control rats, [ 15NIurea protons were observed in 16 min. The HMQC method is likely to be effective for the in vivo study of cerebral and hepatic nitrogen metabolism.

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

    PubMed

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

    2015-06-01

    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 (13)C/(15)N-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 (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-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

  9. 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

  10. 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. PMID:12376667

  11. 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

  12. 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.

  13. 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. PMID:8995843

  14. 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.

  15. Properties of bridgehead-substituted polycycloalkanes. Synthesis and NMR analysis of /sup 15/N-labeled 1-aminobicycloalkanes and their hydrochlorides

    SciTech Connect

    Della, E.W.; Kasum, B.; Kirkbride, K.P.

    1987-04-29

    NMR analysis of adamantane and four bicycloalkanes substituted at the bridgehead with /sup 15/N-labeled amino and ammonio groups is described. It is found that where most of the one-bond carbon-nitrogen coupling constants are relatively large, those in 1-aminobicyclo (1.1.1)pentane and its hydrochloride are significantly reduced; in fact, in the latter compound one-bond /sup 13/C-/sup 15/N coupling could not even be detected. Values of experimentally determined vicinal couplings were in accord with those expected on the basis of the number of three-bond pathways available for transmission of spin-spin information; INDO calculations, however, suggest that in the more highly strained systems there is a substantial contribution to /sup 3/J(CN) arising from through-space interactions and that these oppose through-bond effects. Large four-bond /sup 15/N-/sup 1/H couplings were found to occur in 1-aminobicyclo(1.1.1)pentane and its hydrochloride; MO calculations indicate that through-space interactions constitute the predominant mechanism contributing to /sup 4/J(/sup 15/N-/sup 1/H), although in this case through-bond and through-space effects reinforce each other. The nitrogen-15 chemical shifts of the amine hydrochlorides were determined, and they appear to occur in random fashion.

  16. Vibrational spectra and structure of RDX and its 13C- and 15N-labeled derivatives: a theoretical and experimental study.

    PubMed

    Infante-Castillo, Ricardo; Pacheco-Londoño, Leonardo; Hernández-Rivera, Samuel P

    2010-07-01

    Unambiguous vibrational band assignments have been made to cyclic nitramine hexahydro-1,3,5-trinitro-s-triazine, commonly known as the alpha-phase of RDX or alpha-RDX, with the use of (13)C and (15)N (on ring) enriched isotopic RDX analogues. Vibrational spectra were collected using Raman and IR spectroscopy in solid state and ab initio normal mode calculations were performed using density functional theory (DFT) and a 6-311G++** basis set. The calculated isotopic frequency shifts, induced by (13)C and (15)N labeling, are in very good accordance with measures ones. The changes in vibrational modes associated with the isotopic substitutions are well modeled by the calculation and previous assignments of the vibrational spectra have been revised, especially where the exact nature of the vibrational modes had been either vague or contradictory. PMID:20381411

  17. A new strategy for sequential assignment of intrinsically unstructured proteins based on 15N single isotope labelling

    NASA Astrophysics Data System (ADS)

    Lopez, Juan; Ahuja, Puneet; Gerard, Melanie; Wieruszeski, Jean-Michel; Lippens, Guy

    2013-11-01

    We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional 15N-1H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a Hα-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for α-synuclein.

  18. The 15N isotope effect in Escherichia coli: a neutron can make the difference.

    PubMed

    Filiou, Michaela D; Varadarajulu, Jeeva; Teplytska, Larysa; Reckow, Stefan; Maccarrone, Giuseppina; Turck, Christoph W

    2012-11-01

    Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the (15) N isotope effect on Escherichia coli cultures that were grown in either unlabeled ((14) N) or (15) N-labeled media by LC-ESI-MS/MS-based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between (14) N and (15) N-labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between (14) N and (15) N-labeled bacteria. Our data demonstrate for the first time that the introduction of the (15) N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques. PMID:22887715

  19. Paramagnetic Inversion of the Sign of the Interference Contribution to the Transverse Relaxation of the Imido Protons of the Coordinated Imidazoles in the Uniformly 15N-Labeled Cytochrome c3

    NASA Astrophysics Data System (ADS)

    Ohmura, Tomoaki; Harada, Erisa; Fujiwara, Toshimichi; Kawai, Gota; Watanabe, Kimitsuna; Akutsu, Hideo

    1998-04-01

    In the spectrum of uniformly15N-labeled cytochromec3, the relative linewidths of the doublet peaks of the15N-coupled imido proton of the coordinated imidazole group were reversed on oxidation. This inversion was explained by the interference relaxation process between the electron-proton dipolar and15N-1H dipolar interactions. The inversion can be used to assign the imido protons of the coordinated imidazole groups in heme proteins.

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

    PubMed

    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 (13)C-labeling via the detection and quantification of the (13)C (14)N (-) recombinant ion and the use of the (13)C: (12)C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  1. 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

  2. Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4

    PubMed Central

    Buchinger, Edith; Aachmann, Finn L; Aranko, A Sesilja; Valla, Svein; Skjåk-BræK, Gudmund; Iwaï, Hideo; Wimmer, Reinhard

    2010-01-01

    Alginate epimerases are large multidomain proteins capable of epimerising C5 on β-d-mannuronic acid (M) turning it into α-l-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution-state NMR are hampered by their size—the smallest epimerase, AlgE4, consisting of one A- and one R-module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally 2H, 15N labeled AlgE4 isotopomeres (A-[2H, 15N]-R and [2H, 15N]-A-R) by protein trans-splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4. PMID:20552686

  3. The topology of metabolic isotope labeling networks

    PubMed Central

    Weitzel, Michael; Wiechert, Wolfgang; Nöh, Katharina

    2007-01-01

    Background Metabolic Flux Analysis (MFA) based on isotope labeling experiments (ILEs) is a widely established tool for determining fluxes in metabolic pathways. Isotope labeling networks (ILNs) contain all essential information required to describe the flow of labeled material in an ILE. Whereas recent experimental progress paves the way for high-throughput MFA, large network investigations and exact statistical methods, these developments are still limited by the poor performance of computational routines used for the evaluation and design of ILEs. In this context, the global analysis of ILN topology turns out to be a clue for realizing large speedup factors in all required computational procedures. Results With a strong focus on the speedup of algorithms the topology of ILNs is investigated using graph theoretic concepts and algorithms. A rigorous determination of all cyclic and isomorphic subnetworks, accompanied by the global analysis of ILN connectivity is performed. Particularly, it is proven that ILNs always brake up into a large number of small strongly connected components (SCCs) and, moreover, there are natural isomorphisms between many of these SCCs. All presented techniques are universal, i.e. they do not require special assumptions on the network structure, bidirectionality of fluxes, measurement configuration, or label input. The general results are exemplified with a practically relevant metabolic network which describes the central metabolism of E. coli comprising 10390 isotopomer pools. Conclusion Exploiting the topological features of ILNs leads to a significant speedup of all universal algorithms for ILE evaluation. It is proven in theory and exemplified with the E. coli example that a speedup factor of about 1000 compared to standard algorithms is achieved. This widely opens the door for new high performance algorithms suitable for high throughput applications and large ILNs. Moreover, for the first time the global topological analysis of ILNs

  4. 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.

  5. 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

  6. 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.

  7. Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide 15N–15N and carbonyl 13C–13C dipolar recoupling data

    PubMed Central

    Hu, Kan-Nian; Qiang, Wei; Bermejo, Guillermo A.; Schwieters, Charles D.; Tycko, Robert

    2013-01-01

    Recent structural studies of uniformly 15N, 13C-labeled proteins by solid state nuclear magnetic resonance (NMR) rely principally on two sources of structural restraints: (i) restraints on backbone conformation from isotropic 15N and 13C chemical shifts, based on empirical correlations between chemical shifts and backbone torsion angles; (ii) restraints on inter-residue proximities from qualitative measurements of internuclear dipole–dipole couplings, detected as the presence or absence of inter-residue crosspeaks in multidimensional spectra. We show that site-specific dipole–dipole couplings among 15N-labeled backbone amide sites and among 13C-labeled backbone carbonyl sites can be measured quantitatively in uniformly-labeled proteins, using dipolar recoupling techniques that we call 15N-BARE and 13C-BARE (BAckbone REcoupling), and that the resulting data represent a new source of restraints on backbone conformation. 15N-BARE and 13C-BARE data can be incorporated into structural modeling calculations as potential energy surfaces, which are derived from comparisons between experimental 15N and 13C signal decay curves, extracted from crosspeak intensities in series of two-dimensional spectra, with numerical simulations of the 15N-BARE and 13C-BARE measurements. We demonstrate this approach through experiments on microcrystalline, uniformly 15N, 13C-labeled protein GB1. Results for GB1 show that 15N-BARE and 13C-BARE restraints are complementary to restraints from chemical shifts and inter-residue crosspeaks, improving both the precision and the accuracy of calculated structures. PMID:22449573

  8. H/D exchange of a 15N labelled Tau fragment as measured by a simple Relax-EXSY experiment

    NASA Astrophysics Data System (ADS)

    Lopez, Juan; Ahuja, Puneet; Landrieu, Isabelle; Cantrelle, François-Xavier; Huvent, Isabelle; Lippens, Guy

    2014-12-01

    We present an equilibrium H/D exchange experiment to measure the exchange rates of labile amide protons in intrinsically unfolded proteins. By measuring the contribution of the H/D exchange to the apparent T1 relaxation rates in solvents of different D2O content, we can easily derive the rates of exchange for rapidly exchanging amide protons. The method does not require double isotope labelling, is sensitive, and requires limited fitting of the data. We demonstrate it on a functional fragment of Tau, and provide evidence for the hydrogen bond formation of the phosphate moiety of Ser214 with its own amide proton in the same fragment phosphorylated by the PKA kinase.

  9. An NMR study of the interaction of 15N-labelled bradykinin with an antibody mimic of the bradykinin B2 receptor.

    PubMed

    Ottleben, H; Haasemann, M; Ramachandran, R; Görlach, M; Müller-Esterl, W; Brown, L R

    1997-03-01

    An isotope-edited NMR study of the peptide hormone bradykinin (RPPGFSPFR) bound to the Fab fragment of a monoclonal antibody against bradykinin (MBK3) is reported. MBK3 was previously shown to provide a binding site model of the B2 bradykinin receptor [Haasemann, M., Buschko, J., Faussner, A., Roscher, A. A., Hoebeke, J., Burch, R. M. & Muller-Esterl, W. (1991) Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Immunol. 147, 3882-3892]. Bradykinin was obtained in a uniformly 15N-labelled form using recombinant expression of a fusion protein consisting of the glutathione-binding domain of glutathione S-transferase fused to residues 354-375 of the high-molecular-mass kininogen from which bradykinin was released by proteolytic digestion with its natural protease plasma kallikrein. Bradykinin forms a complex with the Fab fragment of MBK3 which exchanges slowly on the NMR time scale. The 15N and 1H resonances of the tightly bound residues of bradykinin show appreciable changes in chemical shift with respect to the free form, while the 15N and 1H linewidths indicate that the hydrodynamic behaviour of bound bradykinin is dominated by the high-molecular-mass Fab fragment. The NMR data indicate that essentially the entire nonapeptide is involved in binding. The kinetics of the ligand-exchange process, together with resonance assignments obtained via exchange spectroscopy. indicate that bradykinin binds to MBK3 only in the all-trans conformation at all three Xaa-Pro amide bonds. NH-NH NOE connectivities suggest that bradykinin is bound in an extended conformation. The spectroscopic data obtained from this study are compared to recently proposed computational models of the conformation of bradykinin bound to the B2 receptor. PMID:9119014

  10. 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

  11. 15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy.

    PubMed

    Griffey, R H; Davis, D R; Yamaizumi, Z; Nishimura, S; Hawkins, B L; Poulter, C D

    1986-09-15

    Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine

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

    NASA Astrophysics Data System (ADS)

    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 α-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. 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

  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. 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. PMID:21170670

  16. Metabolic labeling of lutropin with [35S]sulfate.

    PubMed Central

    Hortin, G; Natowicz, M; Pierce, J; Baenziger, J; Parsons, T; Boime, I

    1981-01-01

    Chemical analyses have previously detected sulfate linked to the oligosaccharides of lutropin isolated from bovine and human pituitaries. To determine whether lutropin could be metabolically labeled with sulfate, isolated bovine and rat pituitaries were incubated with [35S]sulfate. In both species, two major labeled products were immunoprecipitated with antisera specific to lutropin subunits. Incorporation into the subunits occurred posttranslationally since it was not blocked by cycloheximide, which did, however, block the incorporation of radiolabeled methionine. Metabolic labeling with [35S]sulfate provides a valuable approach for examining the biosynthetic processing of lutropin and the physiological role of sulfate in this hormone. Images PMID:6950389

  17. First results on the incorporation and excretion of 15N from orally administered urea in lactating pony mares.

    PubMed

    Schubert, R; Zander, R; Gruhn, K; Hennig, A

    1991-05-01

    Two lactating pony mares were given oral offers of 20 g 15N urea [95 atom-% 15N-excess (15N')] on 6 subsequent days. About 80% of the consumed 15N' were excreted via urine and faeces, but only about 2% via milk. The 15N' secreted via milk-lysine only amounted to 0.04% of the 15N' intake. The recovery was about 90% in each case. Tissues with active metabolism had an unexpectedly high labelling (greater than 0.3 atom-% 15N'). The low extent of the conversion of oral urea N into milk-lysine speaks against an essential participation of the enteral synthesis in meeting the amino acid requirement of lactating mares. It was already concluded from this results that the determination of the amino acid requirement will be necessary for this group of performance. PMID:1888274

  18. THE EFFECTS OF PARAMAGNETIC RELAXATION REAGENTS ON 15N SPIN RELAXATION AND THE USE OF GD(DPM)3 AS A NITROGEN-15 NUCLEAR MAGNETIC RESONANCE SPIN LABEL

    EPA Science Inventory

    Electron-nuclear relaxation times (T(1) sup e's) for (15)N and (13)C in natural abundance are measured for a series of amines of a wide range of pK(a)s using four paramagnetic relaxation reagents that are soluable in organic solutions. Cr(acac)3 and Cr(dpm)3 are seen to affect th...

  19. In Vitro Metabolic Labeling of Intestinal Microbiota for Quantitative Metaproteomics.

    PubMed

    Zhang, Xu; Ning, Zhibin; Mayne, Janice; Deeke, Shelley A; Li, Jennifer; Starr, Amanda E; Chen, Rui; Singleton, Ruth; Butcher, James; Mack, David R; Stintzi, Alain; Figeys, Daniel

    2016-06-21

    Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-heavy protein/peptide ratio measurements were obtained using a high-resolution mass spectrometer and the quantitative proteomic software tool Census. We subsequently employed our approach to study the in vitro modulating effects of fructo-oligosaccharide and five different monosaccharides on the microbiota. Our methodology improves the accuracy of quantitative intestinal metaproteomics, which would promote the application of proteomics for functional studies of intestinal microbiota. PMID:27248155

  20. High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

    PubMed Central

    Blanchard, Véronique; Gadkari, Rupali A.; George, Albert V. E.; Roy, Satarupa; Gerwig, Gerrit J.; Leeflang, Bas R.; Dighe, Rajan R.; Boelens, Rolf

    2008-01-01

    The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays. PMID:18274893

  1. Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

    SciTech Connect

    Li, Zhou; Adams, Rachel M; Chourey, Karuna; Hurst, Gregory {Greg} B; Hettich, Robert {Bob} L; Pan, Chongle

    2012-01-01

    A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.

  2. Metabolic Labeling in the Study of Mammalian Ribosomal RNA Synthesis.

    PubMed

    Stefanovsky, Victor Y; Moss, Tom

    2016-01-01

    RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates. PMID:27576716

  3. Quantifying plant phenotypes with isotopic labeling & metabolic flux analysis.

    PubMed

    Allen, Doug K

    2016-02-01

    Analyses of metabolic flux using stable isotopes in plants have traditionally been restricted to tissues with presumed homogeneous cell populations and long metabolic steady states such as developing seeds, cell suspensions, or cultured roots and root tips. It is now possible to describe these and other metabolically more dynamic tissues such as leaves in greater detail using novel methods in mass spectrometry, isotope labeling strategies, and transient labeling-based flux analyses. Such studies are necessary for a systems level description of plant function that more closely represents biological reality, and provides insights into the genes that will need to be modified as natural resources become ever more limited and environments change. PMID:26613198

  4. σ-Hole bonding in 15N-labeled N-Benzyl- N-(4-iodo-tetrafluorobenzyl)-amine: Synthesis, crystal structure and solid-state structure calculations

    NASA Astrophysics Data System (ADS)

    Raouafi, Noureddine; Mayer, Peter; Boujlel, Khaled; Schöllhorn, Bernd

    2011-03-01

    Reductive amination of 4-iodo-tetrafluorobenzaldehyde 2 and 15N-enriched benzylamine yielded the title compound 1. Single crystal X-ray diffraction (XRD) revealed that the product crystallizes in the triclinic system of the P-1 space group. The structure is consisting of infinite one-dimensional chair like chains, based on intermolecular N···I halogen bonding. Only intermolecular weak hydrogen bonds N sbnd H···F and C sbnd H···F are observed. Representative XRD data have been compared to the results of theoretical semi-empirical calculations in the solid-state obtained using the PM6 method. Charges of I, N and F atoms are calculated from Natural Bond Orbital (NBO) and Electrostatic Potential Surface maps have been estimated by applying second-order Møller-Plesset (MP2) perturbation theory, and confirmed clearly the assumption of σ-hole bonding formation.

  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. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    SciTech Connect

    O'Grady, J; Schwender, J; Shachar-Hill, Y; Morgan, JA

    2012-03-26

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (CO2)-C-13 dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  7. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    SciTech Connect

    O'Grady J.; Schwender J.; Shachar-Hill, Y.; Morgan, J. A.

    2012-03-01

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on {sup 13}CO{sub 2} dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  8. 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. PMID:11440912

  9. Label-free selection of RNA aptamers for metabolic engineering.

    PubMed

    Hwang, Chuhern; Carothers, James M

    2016-08-15

    RNA aptamers can be assembled into genetic regulatory devices that sense and respond to levels of specific cellular metabolites and thus serve an integral part of designing dynamic control into engineered metabolic pathways. Here, we describe a practical method for generating specific and high affinity aptamers to enable the wider use of in vitro selection and a broader application of aptamers for metabolic engineering. Conventional selection methods involving either radioactive labeling of RNA or the use of label-free methods such as SPR to track aptamer enrichment require resources that are not widely accessible to research groups. We present a label-free selection method that uses small volume spectrophotometers to track RNA enrichment paired with previously characterized affinity chromatography methods. Borrowing techniques used in solid phase peptide synthesis, we present an approach for immobilizing a wide range of metabolites to an amino PEGA matrix. As an illustration, we detail laboratory techniques employed to generate aptamers that bind p-aminophenylalanine, a metabolic precursor for bio-based production of plastics and the pristinamycin family of antibiotics. We focused on the development of methods for ligand immobilization, selection via affinity chromatography, and nucleic acid quantification that can be performed with common laboratory equipment. PMID:27339940

  10. Label-free oxygen-metabolic photoacoustic microscopy in vivo

    PubMed Central

    Yao, Junjie; Maslov, Konstantin I.; Zhang, Yu; Xia, Younan; Wang, Lihong V.

    2011-01-01

    Almost all diseases, especially cancer and diabetes, manifest abnormal oxygen metabolism. Accurately measuring the metabolic rate of oxygen (MRO2) can be helpful for fundamental pathophysiological studies, and even early diagnosis and treatment of disease. Current techniques either lack high resolution or rely on exogenous contrast. Here, we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution to noninvasively quantify MRO2in vivo in absolute units. mPAM is the unique modality for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed. Hyperthermia, cryotherapy, melanoma, and glioblastoma were longitudinally imaged in vivo. Counterintuitively, increased MRO2 does not necessarily cause hypoxia or increase oxygen extraction. In fact, early-stage cancer was found to be hyperoxic despite hypermetabolism. PMID:21806264

  11. Label-free oxygen-metabolic photoacoustic microscopy in vivo

    NASA Astrophysics Data System (ADS)

    Yao, Junjie; Maslov, Konstantin I.; Zhang, Yu; Xia, Younan; Wang, Lihong V.

    2011-07-01

    Almost all diseases, especially cancer and diabetes, manifest abnormal oxygen metabolism. Accurately measuring the metabolic rate of oxygen (MRO2) can be helpful for fundamental pathophysiological studies, and even early diagnosis and treatment of disease. Current techniques either lack high resolution or rely on exogenous contrast. Here, we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution to noninvasively quantify MRO2 in vivo in absolute units. mPAM is the unique modality for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed. Hyperthermia, cryotherapy, melanoma, and glioblastoma were longitudinally imaged in vivo. Counterintuitively, increased MRO2 does not necessarily cause hypoxia or increase oxygen extraction. In fact, early-stage cancer was found to be hyperoxic despite hypermetabolism.

  12. Biosynthetic preparation of L-(/sup 13/C)- and (/sup 15/N)glutamate by Brevibacterium flavum

    SciTech Connect

    Walker, T.E.; London, R.E.

    1987-01-01

    The biosynthesis of isotopically labeled L-glutamic acid by the microorganism Brevibacterium flavum was studied with a variety of carbon-13-enriched precursors. The purpose of this study was twofold: (i) to develop techniques for the efficient preparation of labeled L-glutamate with a variety of useful labeling patterns which can be used for other metabolic studies, and (ii) to better understand the metabolic events leading to label scrambling in these strains. B. flavum, which is used commercially for the production of monosodium glutamate, has the capability of utilizing glucose or acetate as a sole carbon source, and important criterion from the standpoint of developing labeling strategies. Unfortunately, singly labeled glucose precursors lead to excessive isotopic dilution which reduces their usefulness. Studies with (3-/sup 13/C)pyruvate indicate that this problem can in principle be overcome by using labeled three-carbon precursors; however, conditions could not be found which would lead to an acceptable yield of isotopically labeled L-glutamate. In contrast, (1-/sup 13/C)- or (2-/sup 13/C)acetate provides relatively inexpensive, readily available precursors for the production of selectively labeled, high enriched L-glutamate. The preparation of L-(/sup 15/N)glutamate from (/sup 15/N)ammonium sulfate was carried out and is a very effective labeling strategy. Analysis of the isotopic distribution in labeled glutamate provides details about the metabolic pathways in these interesting organisms.

  13. Quantum Dots as Strain- and Metabolism-Specific Microbiological Labels

    PubMed Central

    Kloepfer, J. A.; Mielke, R. E.; Wong, M. S.; Nealson, K. H.; Stucky, G.; Nadeau, J. L.

    2003-01-01

    Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate. PMID:12839801

  14. Quantum dots as strain- and metabolism-specific microbiological labels

    NASA Technical Reports Server (NTRS)

    Kloepfer, J. A.; Mielke, R. E.; Wong, M. S.; Nealson, K. H.; Stucky, G.; Nadeau, J. L.

    2003-01-01

    Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.

  15. [Metabolism of labeled exogenous glucose in fiber flax tissues].

    PubMed

    Chikov, V I; Avvakumova, N Iu; Bakirova, G G; Khamidullina, L A

    2005-01-01

    A labeled glucose solution was introduced into cut fiber flax plants (45-50 cm high) using a special unit under a pressure of 0.1 atm for 30 min, 1, and 2 h. The highest quantities of labeled carbon were revealed in the woody tissue. Sucrose made up a considerable proportion in low molecular weight products of [ [2-14C]-glucose transformation (23.5%). Metabolism of labeled glucose in the leaves exposed to sunlight yielded a set of metabolites similar to products of 14CO2 photoassimilation. In the shade, the pattern of 14C distribution in labeled compounds of the water/alcohol soluble fraction remained similar in mature leaves, while in juvenile leaves, 14C content decreased in sucrose and increased in organic and amino acids. In the shade, the incorporation of 14C into starch and hot water soluble polysaccharides increased at the expense of the acetone fraction (lipids and pigments), water/salt soluble proteins, and cellulose. Low light conditions increased the radioactivity ratio of sparingly soluble (KOH and Triton X-100 soluble) proteins to albumins and globulins. We propose that the synthesis of components of the photosynthetic apparatus in juvenile leaves is directly powered by photosynthesis and the photosynthesis of glucose and the polymers compete for ATP energy. Appearance of sucrose in the woody tissue is due to its release from the phloem to the stem apoplast and the radial transfer to the xylem, where it is transported to the upper shoot with the transpiration flow. PMID:16004260

  16. Generation, Quantification, and Tracing of Metabolically Labeled Fluorescent Exosomes.

    PubMed

    Coscia, Carolina; Parolini, Isabella; Sanchez, Massimo; Biffoni, Mauro; Boussadia, Zaira; Zanetti, Cristiana; Fiani, Maria Luisa; Sargiacomo, Massimo

    2016-01-01

    Over the last 10 years, the constant progression in exosome (Exo)-related studies highlighted the importance of these cell-derived nano-sized vesicles in cell biology and pathophysiology. Functional studies on Exo uptake and intracellular trafficking require accurate quantification to assess sufficient and/or necessary Exo particles quantum able to elicit measurable effects on target cells. We used commercially available BODIPY(®) fatty acid analogues to label a primary melanoma cell line (Me501) that highly and spontaneously secrete nanovesicles. Upon addition to cell culture, BODIPY fatty acids are rapidly incorporated into major phospholipid classes ultimately producing fluorescent Exo as direct result of biogenesis. Our metabolic labeling protocol produced bright fluorescent Exo that can be examined and quantified with conventional non-customized flow cytometry (FC) instruments by exploiting their fluorescent emission rather than light-scattering detection. Furthermore, our methodology permits the measurement of single Exo-associated fluorescence transfer to cells making quantitative the correlation between Exo uptake and activation of cellular processes. Thus the protocol presented here appears as an appropriate tool to who wants to investigate mechanisms of Exo functions in that it allows for direct and rapid characterization and quantification of fluorescent Exo number, intensity, size, and eventually evaluation of their kinetic of uptake/secretion in target cells. PMID:27317184

  17. Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for (13)C metabolic flux analysis.

    PubMed

    Au, Jennifer; Choi, Jungik; Jones, Shawn W; Venkataramanan, Keerthi P; Antoniewicz, Maciek R

    2014-11-01

    In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and (13)C-metabolic flux analysis ((13)C-MFA). Here, cells were grown in parallel cultures with [1-(13)C]glucose and [U-(13)C]glucose as tracers and (13)C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of (13)C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for (13)C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased (13)C-flux measurements in C. acetobutylicum. PMID:25183671

  18. QUANTITATIVE 15N NMR SPECTROSCOPY

    EPA Science Inventory

    Line intensities in 15N NMR spectra are strongly influenced by spin-lattice and spin-spin relaxation times, relaxation mechanisms and experimental conditions. Special care has to be taken in using 15N spectra for quantitative purposes. Quantitative aspects are discussed for the 1...

  19. Theoretical Basis for Dynamic Label Propagation in Stationary Metabolic Networks under Step and Periodic Inputs

    PubMed Central

    Sokol, Serguei; Portais, Jean-Charles

    2015-01-01

    The dynamics of label propagation in a stationary metabolic network during an isotope labeling experiment can provide highly valuable information on the network topology, metabolic fluxes, and on the size of metabolite pools. However, major issues, both in the experimental set-up and in the accompanying numerical methods currently limit the application of this approach. Here, we propose a method to apply novel types of label inputs, sinusoidal or more generally periodic label inputs, to address both the practical and numerical challenges of dynamic labeling experiments. By considering a simple metabolic system, i.e. a linear, non-reversible pathway of arbitrary length, we develop mathematical descriptions of label propagation for both classical and novel label inputs. Theoretical developments and computer simulations show that the application of rectangular periodic pulses has both numerical and practical advantages over other approaches. We applied the strategy to estimate fluxes in a simulated experiment performed on a complex metabolic network (the central carbon metabolism of Escherichia coli), to further demonstrate its value in conditions which are close to those in real experiments. This study provides a theoretical basis for the rational interpretation of label propagation curves in real experiments, and will help identify the strengths, pitfalls and limitations of such experiments. The cases described here can also be used as test cases for more general numerical methods aimed at identifying network topology, analyzing metabolic fluxes or measuring concentrations of metabolites. PMID:26641860

  20. Insights into primary metabolism in oilseeds from labeling and flux analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Labeling investigations along with metabolic flux analysis have enabled quantification of important cellular phenotypes. These descriptions have documented uses of enzymes in unique ways and characterized the contributions of pathways to oil, protein and carbohydrate compositions in seeds. The diffe...

  1. Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

    PubMed Central

    You, Le; Page, Lawrence; Feng, Xueyang; Berla, Bert; Pakrasi, Himadri B.; Tang, Yinjie J.

    2012-01-01

    Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these

  2. Effects of ruminally degradable nitrogen intake and in vitro addition of ammonia and propionate on the metabolic fate of L-[1-14C]alanine and L-[15N]alanine in isolated sheep hepatocytes.

    PubMed

    Mutsvangwa, T; Buchanan-Smith, J G; McBride, B W

    1997-04-01

    Isolated hepatocytes prepared from sheep fed a basal diet (bromegrass hay-corn, 50:50 wt/ wt, as-fed basis) with or without urea were used to determine the effects of added ammonia (as NH4Cl) and propionate on the partitioning of C from 1.25 mM L-[1-14C]alanine between oxidation and gluconeogenesis, and the flux of 15N from 1.25 mM L-[15N]alanine to [14N15N]urea and [15N15N]urea. Hepatocyte suspensions were incubated with NH4Cl (0, .31, .63, and 1.25 mM) and (or) propionate (0, .31, .63, and 1.25 mM) in the presence of either 1.25 mM L-[15N]alanine or 1.25 mM L-alanine plus 18.5 kBq of L-[1-14C]alanine. Feeding dietary urea did not affect [1-14C]alanine oxidation to 14CO2 (P = .601), or its conversion to [14C]glucose (P = .576) by isolated hepatocytes. Increasing in vitro concentrations of NH4Cl and propionate between 0 and 1.25 mM reduced [1-14C]alanine oxidation to 14CO2 (P < .001). Increasing NH4Cl concentration between 0 and 1.25 mM reduced [1-14C]alanine conversion to [14C]glucose in isolated hepatocytes (P < .001), whereas addition of propionate between 0 and 1.25 mM stimulated production of [14C]glucose from [1-14C]alanine (P < .001). Feeding urea did not affect in vitro rates of total urea production (P = .655) but increased the production of [14N15N]urea and [15N15N]urea (P < .05). Addition of NH4Cl increased total urea, [14N15N]urea, and [15N15N]urea production (P < .001), but reduced 15N isotopic enrichments of [14N15N]urea and [15N15N]urea (P < .001). Increasing propionate concentration between 0 and 1.25 mM reduced total urea production (P < .001), but [14N15N]urea and [15N15N]urea production was reduced only at 1.25 mM propionate (P < .001). We conclude that NH3 detoxification by isolated sheep hepatocytes increases amino acid deamination and this might have implications for nitrogen retention in ruminants consuming diets that promote considerable NH3 absorption from the digestive tract. PMID:9110231

  3. In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates

    PubMed Central

    Wang, Hua; Tang, Li; Liu, Yang; Dobrucka, Iwona T.; Dobrucki, Lawrence W.; Yin, Lichen; Cheng, Jianjun

    2016-01-01

    Unnatural sugar-mediated metabolic labeling of cancer cells, coupled with efficient Click chemistry, has shown great potential for in vivo imaging and cancer targeting. Thus far, chemical labeling of cancer cells has been limited to the small-sized azido groups, with the large-sized and highly hydrophobic dibenzocyclooctyne (DBCO) being correspondingly used as the targeting ligand. However, surface modification of nanomedicines with DBCO groups often suffers from low ligand density, difficult functionalization, and impaired physiochemical properties. Here we report the development of DBCO-bearing unnatural sugars that could directly label LS174T colon cancer cells with DBCO groups and subsequently mediate cancer-targeted delivery of azido-modified silica nanoconjugates with easy functionalization and high azido density in vitro and in vivo. This study, for the first time, demonstrates the feasibility of metabolic labeling of cancer cells with large-sized DBCO groups for subsequent, efficient targeting of azido-modified nanomedicines. PMID:27375793

  4. Stable isotope-labeling studies in metabolomics: new insights into structure and dynamics of metabolic networks

    PubMed Central

    Chokkathukalam, Achuthanunni; Kim, Dong-Hyun; Barrett, Michael P; Breitling, Rainer; Creek, Darren J

    2014-01-01

    The rapid emergence of metabolomics has enabled system-wide measurements of metabolites in various organisms. However, advances in the mechanistic understanding of metabolic networks remain limited, as most metabolomics studies cannot routinely provide accurate metabolite identification, absolute quantification and flux measurement. Stable isotope labeling offers opportunities to overcome these limitations. Here we describe some current approaches to stable isotope-labeled metabolomics and provide examples of the significant impact that these studies have had on our understanding of cellular metabolism. Furthermore, we discuss recently developed software solutions for the analysis of stable isotope-labeled metabolomics data and propose the bioinformatics solutions that will pave the way for the broader application and optimal interpretation of system-scale labeling studies in metabolomics. PMID:24568354

  5. GC-MS determination of ratios of stable-isotope labelled to natural urea using [13C15N2]urea for studying urea kinetics in serum and as a means to validate routine methods for the quantitative assay of urea in dialysate.

    PubMed

    Wolthers, B G; Tepper, T; Withag, A; Nagel, G T; de Haan, T H; van Leeuwen, J J; Stegeman, C A; Huisman, R M

    1994-02-01

    A GC-MS determination of urea in serum or spent dialysate is described, using 13C15N2-labelled urea and assaying the area ratio of labelled to natural urea by mass fragmentographic monitoring of fragments m/e 153 and 156, after its eventual conversion into the trimethylsilylether-derivative of 2-hydroxypyrimidine. The procedure can be successfully applied in the follow-up of the disappearance of labelled urea in serum after intravenous injection in man, enabling kinetic parameters of urea to be established, e.g. for purposes of studying the effectiveness of dialysis procedures. Furthermore the method can be used for validation of routine methods for measuring urea in other fluids, in particular dialysate. Examples are given of both applications of the GC-MS method described. PMID:8033352

  6. (15)N NMR studies of a nitrile-modified nucleoside.

    PubMed

    Gillies, Anne T; Gai, Xin Sonia; Buckwalter, Beth L; Fenlon, Edward E; Brewer, Scott H

    2010-12-30

    Nitrile-modified molecules have proven to be excellent probes of local environments in biomolecules via both vibrational and fluorescence spectroscopy. The utility of the nitrile group as a spectroscopic probe has been expanded here to (15)N NMR spectroscopy by selective (15)N incorporation. The (15)N NMR chemical shift (δ((15)N)) of the (15)N-labeled 5-cyano-2'-deoxyuridine (C(15)NdU, 1a) was found to change from 153.47 to 143.80 ppm in going from THF-d(8) to D(2)O. A 0.81 ppm downfield shift was measured upon formation of a hydrogen-bond-mediated heterodimer between 2,6-diheptanamidopyridine and a silyl ether analogue of 1a in chloroform, and the small intrinsic temperature dependence of δ((15)N) of C(15)NdU was measured as a 0.38 ppm downfield shift from 298 to 338 K. The experiments were complemented with density functional theory calculations exploring the effect of solvation on the (15)N NMR chemical shift. PMID:21126044

  7. Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling.

    PubMed

    Palmblad, Magnus; Mills, Davinia J; Bindschedler, Laurence V

    2008-02-01

    We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing. PMID:18189342

  8. Quantifying plant phenotypes with isotopic labeling and metabolic flux analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Analyses of metabolic flux using stable isotopes in plants have traditionally been restricted to tissues with presumed homogeneous cell populations such as developing seeds, cell suspensions, or cultured roots and root tips. It is now possible to describe these and other more complex tissues such a...

  9. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    PubMed Central

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  10. Identification of novel hydrazine metabolites by 15N-NMR.

    PubMed

    Preece, N E; Nicholson, J K; Timbrell, J A

    1991-05-01

    15N-NMR has been used to study the metabolism of hydrazine in rats in vivo. Single doses of [15N2]hydrazine (2.0 mmol/kg: 98.6% g atom) were administered to rats and urine collected for 24 hr over ice. A number of metabolites were detected by 15N-NMR analysis of lyophilized urine. Ammonia was detected as a singlet at 0 ppm and unchanged [15N2]hydrazine was present in the urine detectable as a singlet at 32 ppm. Peaks were observed at 107 and 110 ppm which were identified as being due to the hydrazido nitrogen of acetylhydrazine and diacetylhydrazine, respectively. A resonance at 85 ppm was ascribed to carbazic acid, resulting from reaction of hydrazine with carbon dioxide. A singlet detected at 316 ppm was thought to be due to the hydrazono nitrogen of the pyruvate hydrazone. The resonance at 56 ppm was assigned to 15N-enriched urea, this together with the presence of ammonia indicates that the N-N bond of hydrazine is cleaved in vivo, possibly by N-oxidation, and the resultant ammonia is incorporated into urea. A doublet centred at 150 ppm and a singlet at 294 ppm were assigned to a metabolite which results from cyclization of the 2-oxoglutarate hydrazone. Therefore 15N-NMR spectroscopic analysis of urine has yielded significant new information on the metabolism of hydrazine. PMID:2018564

  11. Comparison of cerebral glucose metabolic rates measured with fluorodeoxyglucose and glucose labeled in the 1, 2, 3-4, and 6 positions using double label quantitative digital autoradiography

    SciTech Connect

    Lear, J.L.; Ackermann, R.F.

    1988-08-01

    We compared local cerebral glucose metabolic rates (LCMRglu) that were determined with (/sup 18/F)fluorodeoxyglucose (FDG) and (/sup 14/C)glucose labeled in the 1, 2, 3-4, and 6 positions. Double label digital autoradiography was used with published kinetic models to determine LCMRglu for FDG and glucose in the same animals. Glucose showed metabolic rate dependent underestimation of LCMRglu compared to FDG, which worsened with increasing experimental times. The least underestimation occurred with glucose labeled in the 6 position at 6 min, reaching 10% in areas of high metabolism. Labeling in the 1 position, the 2 position and the 3-4 position caused progressively worse underestimation at all times. In addition, some structures showed differences not directly related to metabolic rate, indicating regional variations in relationships between individual kinetic constants of FDG and glucose.

  12. Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

    PubMed Central

    Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

    2015-01-01

    The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

  13. 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. PMID:27059012

  14. Metabolic Flux Elucidation for Large-Scale Models Using 13C Labeled Isotopes

    PubMed Central

    Suthers, Patrick F.; Burgard, Anthony P.; Dasika, Madhukar S.; Nowroozi, Farnaz; Van Dien, Stephen; Keasling, Jay D.; Maranas, Costas D.

    2007-01-01

    A key consideration in metabolic engineering is the determination of fluxes of the metabolites within the cell. This determination provides an unambiguous description of metabolism before and/or after engineering interventions. Here, we present a computational framework that combines a constraint-based modeling framework with isotopic label tracing on a large-scale. When cells are fed a growth substrate with certain carbon positions labeled with 13C, the distribution of this label in the intracellular metabolites can be calculated based on the known biochemistry of the participating pathways. Most labeling studies focus on skeletal representations of central metabolism and ignore many flux routes that could contribute to the observed isotopic labeling patterns. In contrast, our approach investigates the importance of carrying out isotopic labeling studies using a more comprehensive reaction network consisting of 350 fluxes and 184 metabolites in Escherichia coli including global metabolite balances on cofactors such as ATP, NADH, and NADPH. The proposed procedure is demonstrated on an E. coli strain engineered to produce amorphadiene, a precursor to the anti-malarial drug artemisinin. The cells were grown in continuous culture on glucose containing 20% [U-13C]glucose; the measurements are made using GC-MS performed on 13 amino acids extracted from the cells. We identify flux distributions for which the calculated labeling patterns agree well with the measurements alluding to the accuracy of the network reconstruction. Furthermore, we explore the robustness of the flux calculations to variability in the experimental MS measurements, as well as highlight the key experimental measurements necessary for flux determination. Finally, we discuss the effect of reducing the model, as well as shed light onto the customization of the developed computational framework to other systems. PMID:17632026

  15. Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

    PubMed Central

    2016-01-01

    Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

  16. Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling.

    PubMed

    Wei, Lu; Shen, Yihui; Xu, Fang; Hu, Fanghao; Harrington, Jamie K; Targoff, Kimara L; Min, Wei

    2015-03-20

    Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse-chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial-temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse-chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

  17. 13C-labelled microdialysis studies of cerebral metabolism in TBI patients☆

    PubMed Central

    Carpenter, Keri L.H.; Jalloh, Ibrahim; Gallagher, Clare N.; Grice, Peter; Howe, Duncan J.; Mason, Andrew; Timofeev, Ivan; Helmy, Adel; Murphy, Michael P.; Menon, David K.; Kirkpatrick, Peter J.; Carpenter, T. Adrian; Sutherland, Garnette R.; Pickard, John D.; Hutchinson, Peter J.

    2014-01-01

    Human brain chemistry is incompletely understood and better methodologies are needed. Traumatic brain injury (TBI) causes metabolic perturbations, one result of which includes increased brain lactate levels. Attention has largely focussed on glycolysis, whereby glucose is converted to pyruvate and lactate, and is proposed to act as an energy source by feeding into neurons’ tricarboxylic acid (TCA) cycle, generating ATP. Also reportedly upregulated by TBI is the pentose phosphate pathway (PPP) that does not generate ATP but produces various molecules that are putatively neuroprotective, antioxidant and reparative, in addition to lactate among the end products. We have developed a novel combination of 13C-labelled cerebral microdialysis both to deliver 13C-labelled substrates into brains of TBI patients and recover the 13C-labelled metabolites, with high-resolution 13C NMR analysis of the microdialysates. This methodology has enabled us to achieve the first direct demonstration in humans that the brain can utilise lactate via the TCA cycle. We are currently using this methodology to make the first direct comparison of glycolysis and the PPP in human brain. In this article, we consider the application of 13C-labelled cerebral microdialysis for studying brain energy metabolism in patients. We set this methodology within the context of metabolic pathways in the brain, and 13C research modalities addressing them. PMID:24361470

  18. Metabolic alkene labeling and in vitro detection of histone acylation via the aqueous oxidative Heck reaction

    PubMed Central

    Ourailidou, Maria E.; Dockerty, Paul; Witte, Martin; Poelarends, Gerrit J.; Dekker, Frank J.

    2016-01-01

    The detection of protein lysine acylations remains a challenge due to a lack of specific antibodies for acylations with various chain lengths. This problem can be addressed by metabolic labeling techniques using carboxylates with reactive functionalities. Subsequent chemoselective reactions with a complementary moiety connected to a detection tag enable the visualization and quantification of the protein lysine acylome. In this study, we present EDTA-Pd(II) as a novel catalyst for the oxidative Heck reaction on protein-bound alkenes, which allows employment of fully aqueous reaction conditions. We used this reaction to monitor histone lysine acylation in vitro after metabolic incorporation of olefinic carboxylates as chemical reporters. PMID:25672493

  19. Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope-assisted labeling.

    PubMed

    Haviland, Julia A; Tonelli, Marco; Haughey, Dermot T; Porter, Warren P; Assadi-Porter, Fariba M

    2012-08-01

    Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, type 2 diabetes mellitus, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy and cavity ring-down spectroscopy to analyze serial plasma samples and real-time breath measurements following selective (13)C-isotope-assisted labeling. These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. This novel diagnostics approach is fast, noninvasive, and sensitive for determining specific pathway utilization, and provides a direct translational application in the health care field. PMID:22304834

  20. Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope assisted labeling (SIAL)

    PubMed Central

    Haviland, Julia A.; Tonelli, Marco; Haughey, Dermot T.; Porter, Warren P.; Assadi-Porter, Fariba M.

    2012-01-01

    OBJECTIVE Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, diabetes mellitus type II, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. METHODS We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy, and cavity ringdown spectroscopy to analyze serial plasma samples and real-time breath measurements following selective 13C-isotope assisted labeling (SIAL). These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. RESULTS Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. CONCLUSION This novel diagnostics approach is fast, non-invasive and sensitive for determining specific pathway utilization, and provides a direct translational application in the healthcare field. PMID:22304834

  1. Microbial metabolism in soil at low temperatures: Mechanisms unraveled by position-specific 13C labeling

    NASA Astrophysics Data System (ADS)

    Bore, Ezekiel

    2016-04-01

    Microbial transformation of organic substances in soil is the most important process of the C cycle. Most of the current studies base their information about transformation of organic substances on incubation studies under laboratory conditions and thus, we have a profound knowledge on SOM transformations at ambient temperatures. However, metabolic pathway activities at low temperature are not well understood, despite the fact that the processes are relevant for many soils globally and seasonally. To analyze microbial metabolism at low soil temperatures, isotopomeres of position-specifically 13C labeled glucose were incubated at three temperature; 5, -5 -20 oC. Soils were sampled after 1, 3 and 10 days and additionally after 30 days for samples at -20 °C. The 13C from individual molecule position was quantifed in respired CO2, bulk soil, extractable organic C and extractable microbial biomass by chloroform fumigation extraction (CFE) and cell membranes of microbial communities classified by 13C phospholipid fatty acid (PLFA) analysis. 13CO2 released showed a dominance of the flux from C-1 position at 5 °C. Consequently, at 5 °C, pentose phosphate pathway activity is a dominant metabolic pathway of glucose metabolization. In contrast to -5 °C and -20 oC, metabolic behaviors completely switched towards a preferential respiration of the glucose C-4 position. With decreasing temperature, microorganism strongly shifted towards metabolization of glucose via glycolysis which indicates a switch to cellular maintenance. High recoveries of 13C in extractable microbial biomass at -5 °C indicates optimal growth condition for the microorganisms. PLFA analysis showed high incorporation of 13C into Gram negative bacteria at 5 °C but decreased with temperature. Gram positive bacteria out-competed Gram negatives with decreasing temperature. This study revealed a remarkable microbial activity at temperatures below 0 °C, differing significantly from that at ambient

  2. A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis

    PubMed Central

    Liechti, G.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y. V.; VanNieuwenhze, M.; Maurelli, A. T.

    2014-01-01

    Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG. PMID:24336210

  3. Measuring (13)C/(15)N chemical shift anisotropy in [(13)C,(15)N] uniformly enriched proteins using CSA amplification.

    PubMed

    Hung, Ivan; Ge, Yuwei; Liu, Xiaoli; Liu, Mali; Li, Conggang; Gan, Zhehong

    2015-11-01

    Extended chemical shift anisotropy amplification (xCSA) is applied for measuring (13)C/(15)N chemical shift anisotropy (CSA) of uniformly labeled proteins under magic-angle spinning (MAS). The amplification sequence consists of a sequence of π-pulses that repetitively interrupt MAS averaging of the CSA interaction. The timing of the pulses is designed to generate amplified spinning sideband manifolds which can be fitted to extract CSA parameters. The (13)C/(13)C homonuclear dipolar interactions are not affected by the π-pulses due to the bilinear nature of the spin operators and are averaged by MAS in the xCSA experiment. These features make the constant evolution-time experiment suitable for measuring CSA of uniformly labeled samples. The incorporation of xCSA with multi-dimensional (13)C/(15)N correlation is demonstrated with a GB1 protein sample as a model system for measuring (13)C/(15)N CSA of all backbone (15)NH, (13)CA and (13)CO sites. PMID:26404770

  4. PET measurement of glucose membrane transport using labeled analogs: Distinction of transport from metabolic processes

    SciTech Connect

    Holden, J.E.; Koeppe, R.A.; Gatley, S.J.

    1984-01-01

    Carrier mediated glucose transport rates across brain capillary and myocardial cell membranes are many times higher than those expected for simple diffusion, and transport regulation can be an important determinant of tissue metabolic status. The authors have investigated the use of glucose analogs and dynamic positron tomography for the non-invasive measurement of unidirectional membrane transport rates. If analog extraction is sufficiently low, transport rates can be inferred directly from fitted kinetic rate constants. Fitting calculations were seen to be sensitive to the difficult to measure rapid components of the arterial input curves, to contributions from blood-borne label in the early data points, and to interference from other chemical forms in cases of significant phosphorylation. This last uncertainty was studied using serial scans of normal brain after venous injection of the well-transported but poorly phosphorylated analog 3-deoxy-3-fluoroglucose. Transport rate constants derived from 4-parameter fits of three hours of data were compared to those derived from 2-parameter fits of the first 12-20 minutes of data. Errors due to trapped label were absorbed primarily into the apparent distribution volume, allowing accurate estimation of transport rate constants from a brief data acquisition period. The study of the distinction of transport from phosphorylation also bears on the important question of the significance of the individual rate constants in the four-parameter fitting of brief dynamic scan sequences in studies of metabolic rate using 2-deoxy-2-fluoroglucose.

  5. Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-((14)C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs.

    PubMed

    Grunwald, Helge; Hargreaves, Patrick; Gebhardt, Klaus; Klauer, Dominique; Serafyn, Arnaud; Schmitt-Hoffmann, Anne; Schleimer, Michael; Schlotterbeck, Goetz; Wind, Mathias

    2013-11-01

    A systematic comparison between two labeling approaches for the investigation of the in vitro metabolic pattern of pharmaceutical drugs was performed by examining the use of (i) radiolabeled drugs analyzed with LC-MS-offline radiodetection and (ii) stable-isotope labeled drugs, used in a defined mixture with the unlabeled drug and analyzed by LC-MS with recognition of the specific isotopic pattern. (14)C was used for the radioisotope-approach and deuterium for the stable-isotope approach. Olanzapine, diclofenac and ketoconazole were chosen as model drugs, as they are commercially available in their non-, radio- and stable-isotope labeled forms. For all three model drugs, liver microsome- and hepatocyte-incubations (both from rat) were performed with various concentrations and incubation times for both, the radio- and the stable-isotope approaches. The metabolic pattern, including structure elucidation of all detected metabolites, was performed independently for all individual compounds and incubations. Subsequently, the metabolic patterns of the radio-, and the stable-isotope approaches were compared. In conclusion, all metabolites found with the radioisotope approach could also be found with the stable-isotope approach. Although the stable-isotope approach does not provide a quantitative result, it can be considered to be a highly suited analytical alternative for early in vitro metabolism investigations, especially when radiolabeled drug analogues are not yet available and quantitative results are not yet necessary. PMID:23933567

  6. {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.

  7. 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.

  8. 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.

  9. Backbone dynamics of barstar: a (15)N NMR relaxation study.

    PubMed

    Sahu, S C; Bhuyan, A K; Majumdar, A; Udgaonkar, J B

    2000-12-01

    Backbone dynamics of uniformly (15)N-labeled barstar have been studied at 32 degrees C, pH 6.7, by using (15)N relaxation data obtained from proton-detected 2D (1)H-(15)N NMR spectroscopy. (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and steady-state heteronuclear (1)H-(15)N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide (15)N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (tau(m)), the generalized order parameter (S(2)), the effective correlation time for internal motions (tau(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D( parallel)/D( perpendicular)) is 0.82 +/- 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, omega(H), and omega(N), where omega(H),(N) are proton and nitrogen Larmor frequencies. The value of tau(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The tau(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 +/- 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2

  10. Metabolic labeling of Ras with tritiated palmitate to monitor palmitoylation and depalmitoylation

    PubMed Central

    Tsai, Frederick D.; Wynne, Joseph P.; Ahearn, Ian M.; Philips, Mark R.

    2014-01-01

    Summary Metabolic labeling with tritiated palmitate is a direct method for monitoring post-translational modification of Ras proteins with this fatty acid. Advances in intensifying screens have allowed for the easy visualization of tritium without the need for extended exposure times. While more energetic radioisotopes are easier to visualize, the lack of commercial source and need for shielding make them more difficult to work with. Since radiolabeled palmitate is directly incorporated into Ras, its loss can be monitored by traditional pulse-chase experiments that cannot be accomplished with the method of acyl exchange chemistry. As such, tritiated palmitate remains a readily accessible and direct method for monitoring the palmitoylation status of Ras proteins under a multitude of conditions. PMID:24470017

  11. Monitoring Protein O-GlcNAc Status via Metabolic Labeling and Copper-free Click Chemistry

    PubMed Central

    Teo, Chin Fen; Wells, Lance

    2014-01-01

    O-GlcNAc modification found on the serine and threonine residues of intracellular proteins is an inducible post-translational modification that regulates numerous biological processes. In combination with other cell biological and biochemical approaches, a robust and streamlined strategy for detecting the number and stoichiometry of O-GlcNAc modification can provide valuable insights for decoding the functions of O-GlcNAc at the molecular level. Herein, we report an optimized workflow for evaluating the O-GlcNAc status of proteins using a combination of metabolic labeling and click chemistry based mass tagging. This method is strategically complementary to the chemoenzymatic-based mass-tagging method. PMID:24995865

  12. Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods*

    PubMed Central

    Wood, David W.; Camarero, Julio A.

    2014-01-01

    The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification. PMID:24700459

  13. Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging

    PubMed Central

    Wainman, Yéléna A.; Wright, Alan; Kettunen, Mikko I.; Rodrigues, Tiago B.; McGuire, Sarah; Hu, De‐En; Bulat, Flaviu; Geninatti Crich, Simonetta; Stöckmann, Henning; Leeper, Finian J.; Brindle, Kevin M.

    2015-01-01

    Abstract Glycosylation is a ubiquitous post‐translational modification, present in over 50 % of the proteins in the human genome,1 with important roles in cell–cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases,2 including cancer.3 We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium‐based bioorthogonal MRI probe. Significant N‐azidoacetylgalactosamine dependent T 1 contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10‐fold). This approach has the potential to enable the rapid and non‐invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease.

  14. 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.

  15. Effect of metabolism on retention of indium-111-labeled monoclonal antibody in liver and blood

    SciTech Connect

    Kinuyfa, S.; Jeong, J.M.; Garmestani, K.

    1994-11-01

    The effect of a chelator structure on the metabolic fate of the {sup 111}In-labeled monoclonal antibody (Mab) T101 was investigated in normal Balb/c mice to assess the importance of this chemical parameter in the reduction of the background radioactivity in blood and liver. Mab T101 was conjugated with either 2-(p-isothiocyanatobenzyl)-6-methyl-diethylaminetriaminepentaacetic acid (DTPA) (1B4M), 2-(p-isothiocyanatobenzyl) cyclohexyl-DTPA (CHX-B) or cyclic DTPA dianhydride (cDTPA) and then radiolabeled with {sup 111}In-labeled T101 conjugates and sacrificed in groups of five up to 5 days postinjection for comparative biodistribution studies and analyses of liver, blood and urine samples for radioindium products. The biodistribution of {sup 111}In-1B4M-T101 and {sup 111}In-CHX-B-T101 were similar to each other but significantly different from that of {sup 111}In-cDTPA-T101, particularly in the blood and liver. Size-exclusion high-performance liquid chromatography indicated that the concentration of the intact {sup 111}In-immunoglobulin (Ig)G in liver decreased with similar rates for the three conjugates. Meanwhile, the concentration of a small DTPA-like metabolite in liver increased to a different peak value (4.6% 1D/g for the cDTPA conjugate and 1.6% lD/g for the 1B4M and CHX-B conjugates) approximately at 24 hr and maintained a steady-state concentration up to 5 days. The thiourea linkage between T101 and the {sup 111}In-labeled chelates and a higher complex stability and higher lipophilicity of {sup 111}In-1B4M and {sup 111}In-CHX-B appear to be responsible for lower liver and higher blood radioactivity for the 1B4M and CHX conjugates. 31 refs., 3 figs., 1 tab.

  16. Metabolism of glutamine and glutamate in monkey lenses

    SciTech Connect

    Jernigan, H.M. Jr.; Zigler, J.S. Jr.

    1986-05-01

    In rat lenses, glutamine (GLN), not glutamate (GLU), from the surrounding fluids is the primary source of GLU utilized by several metabolic pathways. To study lenticular amino acid metabolism in a primate, fresh lenses from young (2-3 yr) rhesus monkeys (Macaca mulatta) were incubated at 37/sup 0/C for 3, 6, or 24 hr in balanced salt medium containing 5 mM of amino-labeled /sup 15/N-GLN or /sup 15/N-GLU. The % enrichment of /sup 15/N in several free amino acids was determined by GCMS. GLN entered the monkey lenses more rapidly than GLU, but, in contrast to rat lenses, /sup 15/N-GLN did not more rapidly label other amino acids. The % of /sup 15/N in the (GLN + GLU) pool of the monkey lenses in /sup 15/N-GLN reached 20, 35, and 60% at 3, 6, and 24 hr respectively, compared with 10, 20, and 40% in the lenses in /sup 15/N-GLU. However, in monkey lenses incubated 24 hr with /sup 15/N-GLN, the /sup 15/N in alanine, serine, proline, and (aspartate + asparagine) was only 35, 6, 7, and 30% respectively, compared with 50, 10, 7, and 50% in monkey lenses with /sup 15/N-GLU. Compared with rat lenses, monkey lenses showed slower transport, deamidation, and metabolism of GLN, and less serine, proline, and glycine synthesis. Also, part of the GLU in monkey lenses appeared to be in a slowly transaminating pool. Species differences should be considered when rats are used as a model to study changes in human lenses during aging and cataractogenesis.

  17. Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

    PubMed Central

    Berger, Michael G.; Woo, K. C.; Wong, Suan-Chin; Fock, Heinrich P.

    1985-01-01

    Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO2 assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in 15N-labeled NH3, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The 15N-enrichment of various amino acids derived from these 15N-substrates were examined. The amido-N of glutamine was the first 15N-labeled product in leaves incubated with 15NH4Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly 15N-labeled products during incubation with [15N]glycine. In contrast, aspartate and alanine were the first 15N-labeled products when [15N] glutamate was used. These results indicate that NH3 was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues. PMID:16664324

  18. Monitoring electron donor metabolism under variable electron acceptor conditions using 13C-labeled lactate

    NASA Astrophysics Data System (ADS)

    Bill, M.; Conrad, M. E.; Yang, L.; Beller, H. R.; Brodie, E. L.

    2010-12-01

    Three sets of flow-through columns constructed with aquifer sediment from Hanford (WA) were used to study reduction of Cr(VI) to poorly soluble Cr(III) under denitrifying, sulfate-reducing/fermentative, and iron-reducing conditions with lactate as the electron donor. In order to understand the relationship between electron donors and biomarkers, and to determine the differences in carbon isotope fractionation resulting from different microbial metabolic processes, we monitored the variation in carbon isotopes in dissolved inorganic carbon (DIC), in total organic carbon (TOC), and in lactate, acetate and propionate. The greatest enrichment in 13C in columns was observed under denitrifying conditions. The δ13C of DIC increased by ~1750 to ~2000‰ fifteen days after supplementation of natural abundance lactate with a 13C-labeled lactate tracer (for an influent δ13C of ~2250‰ for the lactate) indicating almost complete oxidation of the electron donor. The denitrifying columns were among the most active columns and had the highest cell counts and the denitrification rate was highly correlated with Cr(VI) reduction rate. δ13C values of DIC ranged from ~540 to ~1170‰ for iron-reducing conditions. The lower enrichment in iron columns was related to the lower biological activity observed with lower yields of RNA and cell numbers in the column effluents. The carbon isotope shift in the sulfate-reducing ~198 to ~1960‰ for sulfate-reducing conditions reflecting the lower levels of the lactate in these columns. Additionally, in two of the sulfate columns, almost complete fermentation of the lactate occurred, producing acetate and propionate with the labeled carbon signature, but relatively smaller amounts of inorganic carbon. For all electron-accepting conditions, TOC yielded similar δ13C values as lactate stock solutions. Differences in C use efficiency, metabolic rate or metabolic pathway contributed to the differing TOC δ13C to DIC δ13C ratios between treatments

  19. (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. PMID:26367322

  20. Complete fusion of 15N+27Al

    NASA Astrophysics Data System (ADS)

    Prosser, F. W., Jr.; Racca, R. A.; Daneshvar, K.; Geesaman, D. F.; Henning, W.; Kovar, D. G.; Rehm, K. E.; Tabor, S. L.

    1980-05-01

    The total fusion cross section for the system 15N + 27Al has been measured over an energy range 27 MeV<=Elab<=70 MeV by detection of the fusion-evaporation residues. In addition elastic scattering was measured at six energies and fitted by optical model calculations. The fusion cross section for the system saturates at 1150+/-50 mb. The data can be well described by the model of Glas and Mosel, using a reasonable set of parameters. The model of Horn and Ferguson also describes the data well if an appropriate charge radius is used. Comparison is made between these results and the fusion cross sections for 16O + 26Mg and 18O + 24Mg, which lead to the same compound nucleus. The results for 15N + 27Al are quite similar to those for 18O + 24Mg, and the differences between the fusion cross sections for these two systems and those for 16O + 26Mg may be evidence for an entrance channel effect. NUCLEAR REACTIONS 15N+27Al, Elab=27-70 MeV; measured σfusion(E) measured dσdΩ elastic scattering; data fitted with Glas and Mosel model, Horn and Ferguson model.

  1. Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS▿ †

    PubMed Central

    Behrens, Sebastian; Lösekann, Tina; Pett-Ridge, Jennifer; Weber, Peter K.; Ng, Wing-On; Stevenson, Bradley S.; Hutcheon, Ian D.; Relman, David A.; Spormann, Alfred M.

    2008-01-01

    To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with 13C-carbon and 15N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities. PMID:18359832

  2. Probing the Metabolic Network in Bloodstream-Form Trypanosoma brucei Using Untargeted Metabolomics with Stable Isotope Labelled Glucose

    PubMed Central

    Creek, Darren J.; Mazet, Muriel; Achcar, Fiona; Anderson, Jana; Kim, Dong-Hyun; Kamour, Ruwida; Morand, Pauline; Millerioux, Yoann; Biran, Marc; Kerkhoven, Eduard J.; Chokkathukalam, Achuthanunni; Weidt, Stefan K.; Burgess, Karl E. V.; Breitling, Rainer; Watson, David G.; Bringaud, Frédéric; Barrett, Michael P.

    2015-01-01

    Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate. PMID:25775470

  3. 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

  4. 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.

  5. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Datta, Rupsa; Heylman, Christopher; George, Steven C.; Gratton, Enrico

    2016-01-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  6. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Datta, Rupsa; Heylman, Christopher; George, Steven C; Gratton, Enrico

    2016-05-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  7. Proteomic Analysis of Protein Turnover by Metabolic Whole Rodent Pulse-Chase Isotopic Labeling and Shotgun Mass Spectrometry Analysis.

    PubMed

    Savas, Jeffrey N; Park, Sung Kyu; Yates, John R

    2016-01-01

    The analysis of protein half-life and degradation dynamics has proven critically important to our understanding of a broad and diverse set of biological conditions ranging from cancer to neurodegeneration. Historically these protein turnover measures have been performed in cells by monitoring protein levels after "pulse" labeling of newly synthesized proteins and subsequent chase periods. Comparing the level of labeled protein remaining as a function of time to the initial level reveals the protein's half-life. In this method we provide a detailed description of the workflow required for the determination of protein turnover rates on a whole proteome scale in vivo. Our approach starts with the metabolic labeling of whole rodents by restricting all the nitrogen in their diet to exclusively nitrogen-15 in the form of spirulina algae. After near complete organismal labeling with nitrogen-15, the rodents are then switched to a normal nitrogen-14 rich diet for time periods of days to years. Tissues are harvested, the extracts are fractionated, and the proteins are digested to peptides. Peptides are separated by multidimensional liquid chromatography and analyzed by high resolution orbitrap mass spectrometry (MS). The nitrogen-15 containing proteins are then identified and measured by the bioinformatic proteome analysis tools Sequest, DTASelect2, and Census. In this way, our metabolic pulse-chase approach reveals in vivo protein decay rates proteome-wide. PMID:26867752

  8. Continuous field measurement of N2O isotopologues using FTIR spectroscopy following 15N addition

    NASA Astrophysics Data System (ADS)

    Phillips, R. L.; Griffith, D. W.; Dijkstra, F. A.; Lugg, G.; Lawrie, R.; Macdonald, B.

    2012-12-01

    Anthropogenic additions of fertilizer nitrogen (N) have significantly increased the mole fraction of nitrous oxide (N2O) in the troposphere. Tracking the fate of fertilizer N and its transformation to N2O is important to advance knowledge of greenhouse gas emissions from soils. Transport and transformations are frequently studied using 15N labeling experiments, but instruments capable of continuous measurements of 15N-N2O at the surface of soil have only recently come to the fore. Our primary aim was to quantify emissions of N2O and the fraction of 15N emitted as N2O from an agricultural soil following 15N addition using a mobile Fourier Transform Infrared (FTIR) spectrometer. We set up a short-term field experiment on a coastal floodplain site near Nowra, New South Wales. We deployed an automated chamber system connected to a multi-pass cell (optical pathlength 24 m) and low resolution FTIR spectrometer to measure fluxes of all N2O isotopologues collected from five 0.25 m2 chambers every three hours. We measured N2O fluxes pre and post-application of 15N-labeled substrate as potassium nitrate (KNO3) or urea [CO(NH2)2] to the soil surface. Root mean square uncertainties for all isotopologue measurements were less than 0.3 nmol mol-1 for 1 minute average concentration measurements, and minimum detectable fluxes for each isotopologue were <0.1 ng N m-2 s-1. Emissions of all N2O isotopologues were evident immediately following 15N addition. Emissions of 14N15NO, 15N14NO and 15N15NO isotopologues subsided within 10 d, but 14N14NO fluxes were evident over the entire experiment. The figure provides an overview of the emissions. Cumulative 15N-N2O fluxes (sum of the three 15N isotopologues) per chamber for the 14 days following 15N addition ranged from 1.5 to 10.3 mg 15N-N2O m-2. The chambers were destructively sampled after 2 weeks and 15N analyzed in soil and plant material using isotope ratio mass spectrometry. Approximately 1% (range 0.7 - 1.9%) of the total amount of

  9. Metabolism of pyridalyl in rats: excretion, distribution, and biotransformation of dichloropropenyl-labeled pyridalyl.

    PubMed

    Nagahori, Hirohisa; Tomigahara, Yoshitaka; Isobe, Naohiko; Kaneko, Hideo

    2009-11-25

    Metabolism of pyridalyl [2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether] labeled at position 2 of the dichloropropenyl group with 14C was investigated after single oral administration to male and female rats at 5 and 500 mg/kg. Absorbed 14C was excreted into feces (68-79%), urine (8-14%), and expired air (6-10%) in all of the groups. Regarding 14C-tissue residues on the seventh day after administration, fat showed the highest levels at 0.98-2.34 ppm and 219-221 ppm with the low and high doses, respectively. 14C-Residues in other tissues accounted for 0.03-0.32 ppm at the low dose and 3-70 ppm at the high dose. The percentages of the 14C-residue in fat were 1.50-3.16% of the dose, and those of muscle and hair and skin were also relatively high, accounting for 0.49-1.20%. Total 14C-residues in the whole body were 2.95-6.80% of the dose. Fecal metabolites in male rats treated with 500 mg/kg pyridalyl were purified by a combination of chromatographic techniques, and chemical structures of 8 metabolites were identified by NMR and MS spectrometry. The biotransformation reactions in rats were proposed to be as follows: (1) epoxidation of the double bond in the dichloropropenyl group followed by hydration, dehydrochlorination, decarboxylation, and/or mercapturic acid conjugation; (2) CO2 formation after O-dealkylation of pyridalyl and its metabolites; (3) hydroxylation of C3 in the pyridyl ring; (4) O-dealkylation of the pyridyloxy and the trimethylene groups; (5) dehydrochlorination and hydration in the dichloropropenyl group. PMID:19919122

  10. Metabolic fate of tritium-labeled T-2 toxin, a trichothecene mycotoxin, in swine

    SciTech Connect

    Corley, R.A.

    1985-01-01

    The metabolic fate of T-2 toxin was determined in two female crossbred swine following the intravascular administration of one millicurie of tritium-labeled T-2 toxin at a nonlethal dose of 0.15 mg/kg body weight. The plasma elimination phase half-life was 90 minutes for total tritium residues. A total of 13.1 and 1.3 percent of the administration dose was found in the gall bladders in addition to 17.9 and 42.5% in the urine in the two pigs, S1 and S2, respectively, 4 hours after dosing. Free metabolites, identified by thin-layer chromatography, represented less than 20 and 30% of the metabolite residues in bile and urine, respectively, with the parent compound, T-2 toxin, never exceeding 0.25%. The major free metabolites were 3-OH HT-2 and T-2 triol. Glucuronide conjugates represented 63 and 77% of the metabolite residues in urine and bile, respectively. The major conjugated metabolites were glucuronides of HT-2, 3'-OH T-2, 3'-OH HT-2 and T-2 toxin. Neosolaniol, 4-deacetyl-neosolaniol and T-2 tetraol were also identified in addition to 3 unknown metabolites. In the tissues, the greatest amount of radioactivity was located in the gastrointestinal tract (15.5 and 24.1% of the dose for the 2 pigs, S1 and S2, respectively). The remaining tissues sampled accounted for approximately 5% of the dose for the 2 pigs. Twenty-one metabolites were identified in tissues following reverse phase HPLC radiochromatography.

  11. Metabolic network analysis of Bacillus clausii on minimal and semirich medium using (13)C-labeled glucose.

    PubMed

    Christiansen, Torben; Christensen, Bjarke; Nielsen, Jens

    2002-04-01

    Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium. The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations. It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose. In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium. Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose. The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium. PMID:12009795

  12. 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.

  13. Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

    PubMed Central

    Chikayama, Eisuke; Suto, Michitaka; Nishihara, Takashi; Shinozaki, Kazuo; Hirayama, Takashi; Kikuchi, Jun

    2008-01-01

    Background Metabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems. Methodology/Principal Findings The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 1H and 13C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each 13C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient 13C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts. Conclusions/Significance Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of 13C atoms of

  14. 13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification.

    PubMed

    Hollocher, T C; Garber, E; Cooper, A J; Reiman, R E

    1980-06-10

    13N- and 15N-labeling experiments were carried out with Paracoccus denitrificans, grown anaerobically on nitrate, to determine whether hyponitrite might be an obligatory intermediate in denitrification and a precursor of nitrous oxide. From experiments designed to trap [13N]- or [15N,15N]hyponitrite by dilution into authentic hyponitrite it was calculated that the intracellular concentration of a presumptive hyponitrite pool must be less than 0.4 mM. In order for a pool of this size to turn over rapidly enough to handle the flux of nitrogen during dentrifucation, the spontaneous rate of hyponitrite dehydration must be enhanced by a factor of several thousand through enzyme catalysis. Cell extracts failed to catalyze this reaction under a variety of conditions. It is concluded that hyponitrite cannot be an intermediate in dentrification. In addition, the assimilation of inorganic nitrogen was studied in P. denitrificans using 13N as tracer. At low concentrations (less than 10(-8) M) of labeled nitrate and nitrite 5 to 10% of the label was assimilated into non-volatile metabolites and 90 to 95% was reduced to N2. Similarly, with 15 mM [13N]nitrate, 5% of the label went into metabolites and 95% to N2. High pressure liquid chromatography analysis of the labeled metabolites indicated that the major pathway for assimilation of inorganic nitrogen in P. denitrificans under these conditions is through ammonia incorporation via the aspartase reaction. PMID:7372623

  15. Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

    PubMed Central

    2014-01-01

    Background Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation. Results Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism. Conclusions The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids. PMID:24773761

  16. Application of unsymmetrical indirect covariance NMR methods to the computation of the (13)C <--> (15)N HSQC-IMPEACH and (13)C <--> (15)N HMBC-IMPEACH correlation spectra.

    PubMed

    Martin, Gary E; Hilton, Bruce D; Irish, Patrick A; Blinov, Kirill A; Williams, Antony J

    2007-10-01

    Utilization of long-range (1)H--(15)N heteronuclear chemical shift correlation has continually grown in importance since the first applications were reported in 1995. More recently, indirect covariance NMR methods have been introduced followed by the development of unsymmetrical indirect covariance processing methods. The latter technique has been shown to allow the calculation of hyphenated 2D NMR data matrices from more readily acquired nonhyphenated 2D NMR spectra. We recently reported the use of unsymmetrical indirect covariance processing to combine (1)H--(13)C GHSQC and (1)H--(15)N GHMBC long-range spectra to yield a (13)C--(15)N HSQC-HMBC chemical shift correlation spectrum that could not be acquired in a reasonable period of time without resorting to (15)N-labeled molecules. We now report the unsymmetrical indirect covariance processing of (1)H--(13)C GHMBC and (1)H--(15)N IMPEACH spectra to afford a (13)C--(15)N HMBC-IMPEACH spectrum that has the potential to span as many as six to eight bonds. Correlations for carbon resonances long-range coupled to a protonated carbon in the (1)H--(13)C HMBC spectrum are transferred via the long-range (1)H--(15)N coupling pathway in the (1)H--(15)N IMPEACH spectrum to afford a much broader range of correlation possibilities in the (13)C--(15)N HMBC-IMPEACH correlation spectrum. The indole alkaloid vincamine is used as a model compound to illustrate the application of the method. PMID:17729230

  17. Evaluation of myocardial metabolism, with /sup 13/N- and /sup 11/C-labeled amino acids and positron computed tomography

    SciTech Connect

    Henze, E.; Schelbert, H.R.; Barrio, J.R.; Egbert, J.E.; Hansen, H.W.; MacDonald, N.S.; Phelps, M.E.

    1982-08-01

    To evaluate the utility of labeled L-amino acids (AA) for imaging regional myocardial AA metabolism by positron computed tomography (PCT), the myocardial uptake and clearance of Ala,* Glu, Gln, Asp, Leu tagged with /sup 13/N, and of /sup 11/C-tagged Asp, and oxaloacetate (Oxal), were examined in 44 experiments at control, during ischemia, and after transaminase inhibition. The myocardial time-activity curves recorded after intracoronary tracer injection had two clearance phases (an early and a late) for all /sup 13/N AA, and three (early, intermediate, late) for the two /sup 11/C compounds, with significantly different clearance half-times of 18.7 +/- 8.0 (s.d.) sec for the early phase, 141.7 +/- 56.5 sec for the intermediate, and 61.2 +/- 43.5 min for the late phase. The residual fractions ranged from 0.07 to 0.23 in normal myocardium, and consistently increased with ischemia by 0.01-0.07 for /sup 13/N-labeled Ala, Glu, Asp, and Leu, but not for /sup 13/N Gln and /sup 11/C compounds. Transaminase inhibition shortened the half-times of the late phases of /sup 13/N-labeled Ala, Glu, Asp, and Leu; had no effect on t1/2 of /sup 13/N Gln and /sup 11/C Oxal; and resulted in a loss of /sup 11/C CO/sub 2/ production and of the intermediate phase for /sup 11/C Asp. On the PCT images, /sup 13/N activity from labeled Ala and Glu was not decreased in an ischemic segment despite a significant flow reduction, as demonstrated by /sup 13/N NH/sub 3/ imaging and labeled microspheres. From the results, a three-compartment tracer kinetic model is proposed for the noninvasive quantification of Krebscycle activity, protein synthesis, and metabolic derangements related to ischemia.

  18. Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study.

    PubMed

    Bindschedler, Laurence V; Palmblad, Magnus; Cramer, Rainer

    2008-07-01

    Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as (15)N. By utilising hydroponic media that contain (15)N inorganic salts as the sole nitrogen source, near to 100% (15)N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled (14)N- and (15)N-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of (14)N/(15)N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the (14)N and (15)N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct (14)N and (15)N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2). PMID:18538804

  19. 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.

  20. Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution1[OPEN

    PubMed Central

    Päpke, Carola; Diab, Houssein; Fernie, Alisdair R.

    2016-01-01

    Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [13C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [13C]glutamate and [15N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the 13C/15N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia. PMID:26553649

  1. Label-free proteomics uncovers energy metabolism and focal adhesion regulations responsive for endometrium receptivity.

    PubMed

    Chen, Qian; Zhang, Aijun; Yu, Feng; Gao, Jing; Liu, Yue; Yu, Chengli; Zhou, Hu; Xu, Chen

    2015-04-01

    The menstrual cycle of the female uterus leads to periodic changes of the endometrium. These changes are important for developing the endometrial receptivity and for achieving competency of embryo implantation. However, the molecular events underlying the endometrial receptivity process remain poorly understood. Here we applied an LC-MS-based label-free quantitative proteomic approach to compare the endometrial tissues in the midsecretory (receptive) phase with the endometrial tissues in the proliferative phase from age-matched woman (n = 6/group). The proteomes of endometrial tissues were extracted using an SDS-based detergent, digested by the filter-aided sample preparation procedures, and subsequently analyzed by nano-LC-MS/MS (Orbitrap XL) with a 4 h gradient. Reliable protein expression profiles were reproducibly obtained from the endometrial tissues in the receptive and proliferative phases. A total of 2138 protein groups were quantified under highly stringent criteria with a false discovery rate of <1% for peptide and protein groups. Among these proteins, 317 proteins had differences in expression that were statistically significant between the receptive and proliferative phases. Direct protein-protein interaction network analyses of these significantly changed proteins showed that the up-regulation of creatine kinase B-type (CKB) in the receptive phase may be related to endometrium receptivity. The interaction network also showed that proteins related to cell-cell adhesion were down-regulated. Moreover, the results from KEGG pathway analyses are consistent with the protein-protein interaction results. The proteins, including alpha-actinin (ACTN), extracellular matrix proteins, integrin alpha-V, and so on, that are involved in the focal adhesion pathway were down-regulated in the receptive phase compared with the proliferative phase, which may facilitate the implantation of the fertilized ovum. Selected proteins were validated by Western blot analysis and

  2. Metabolic fate of the (14)C-labeled herbicide clodinafop-propargyl in soil.

    PubMed

    Jaquet, Jasmin; Weitzel, Pascal; Junge, Thorsten; Schmidt, Burkhard

    2014-01-01

    The metabolic fate of (14)C-phenyl-labeled herbicide clodinafop-propargyl (CfP) was studied for 28 days in lab assays using a soil from Germany (Ap horizon, silt loam, and cambisol). Mineralization amounted to 12.40% of applied (14)C after 28 days showing a distinct lag phase until day 7 of incubation. Portions of radioactivity extractable by means of 0.01 M CaCl2 solution (bioavailable fraction) decreased rapidly and were 4.41% after 28 days. Even immediately after application, only 57.31% were extracted with the aqueous solvent. Subsequent extraction using accelerated solvent extraction (ASE; acetonitrile/water 4:1, v/v) released 39.91% of applied (14)C with day 0 and 26.16% with day 28 of incubation from the samples. Non-extractable portions of radioactivity thus, increased with time amounting to 11.99% (day 0) and 65.00% (day 28). A remarkable increase was observed between 14 and 28 days correlating with the distinct increase of mineralization. No correlation was found throughout incubation with general microbial activity as determined by DMSO reduction. Analysis of the CaCl2 and ASE extracts by radio-TLC, radio-HPLC and GC/MS revealed that CfP was rapidly cleaved to free acid clodinafop (Cf), which was further (bio-) transformed; DT50 values (based on radio-TLC detection of the parent compound) were far below 1 day (CfP) and about 7 days (Cf). TLC analysis pointed to 2-(4-hydroxyphenoxy)-propionic acid as further metabolite. Due to fractionation of non-extractable residues, most of the (14)C was associated with fulvic and humic acids, portions in humin fractions and non-humics were moderate and low, respectively. Using a special strategy, which included pre-incubation of the soil with CfP and then mineralization of (14)C-CfP as criterion, a microorganism was isolated from the soil examined. The microorganism grew using CfP as sole carbon source with concomitant evolution of (14)CO2. The bacterium was characterized by growth on commonly used carbon

  3. The Metabolic Fate of Tritium-Labeled T-2 Toxin, a Trichothecene Mycotoxin, in Swine.

    NASA Astrophysics Data System (ADS)

    Corley, Richard Allen

    The metabolic fate of T-2 toxin was determined in two female crossbred swine following the intravascular administration of one millicurie of tritium-labeled T-2 toxin at a nonlethal dose of 0.15 mg/kg body weight. The plasma elimination phase half-life was 90 minutes for total tritium residues. A total of 13.1 and 1.3 percent of the administered dose was found in the gall bladders in addition to 17.9 and 42.5 percent in the urine of the two pigs, S1 and S2, respectively, 4 hours after dosing. Free metabolites, identified by thin-layer chromatography, represented less than 20 and 30 percent of the metabolite residues in bile and urine, respectively, with the parent compound, T-2 toxin, never exceeding 0.25 percent. The major free metabolites were 3'-OH HT-2 and T-2 triol. Glucuronide conjugates represented 63 and 77 percent of the metabolite residues in urine and bile, respectively. The major conjugated metabolites were glucuronides of HT-2, 3'-OH T-2, 3'-OH HT-2 and T-2 toxin. Neosolaniol, 4-deacetyl-neosolaniol and T-2 tetraol were also identified in addition to 3 unknown metabolites. In the tissues, the greatest amount of radioactivity was located in the gastrointestinal tract (15.5 and 24.1 percent of the dose for the 2 pigs, S1 and S2, respectively). The remaining tissues sampled accounted for approximately 5 percent of the dose for the 2 pigs. Twenty-one metabolites were identified in tissues following reverse phase HPLC radiochromatography. Approximately 55 percent of the extractable radioactivity in the tissues, including the gastrointestinal tract, of both pigs corresponded to T-2 toxin, HT-2, deepoxy HT-2, T-2 triol, deepoxy T-2 triol, 3'-OH T-2, 3'-OH HT -2, T-2 tetraol and deepoxy T-2 tetraol. The major metabolite in tissues, PM-XV, did not correspond to any standard and represented an additional 27 percent of the extractable radioactivity.

  4. Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans

    PubMed Central

    Fredens, Julius; Færgeman, Nils J.

    2012-01-01

    Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology to quantitatively examine metabolic and signaling pathways in yeast, fruit flies, plants, cell cultures and mice. However, only metabolic labeling using 15N has been applied to examine such events in the nematode Caenorhabditis elegans. We have recently shown that C. elegans can be completely labeled with heavy-labeled lysine by feeding worms on prelabeled lysine auxotroph Escherichia coli for just one generation. We applied this methodology to examine the organismal response to functional loss or RNAi mediated knock down of the transcription factor NHR-49, and found numerous proteins involved in lipid metabolism to be downregulated, which is consistent with its previously proposed function as a transcriptional regulator of fatty acid metabolism. The combined use of quantitative proteomics and selective gene knockdown by RNAi provides a powerful tool with broad implications for C. elegans biology. PMID:24058826

  5. Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion.

    PubMed

    Oum, Yoon Hyeun; Desai, Tanay M; Marin, Mariana; Melikyan, Gregory B

    2016-07-01

    Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels into the viral membrane and trapping fluorescent proteins in the virus interior enables the visualization of single virus fusion in living cells. Here, we implement a new approach to non-invasively label the viral membrane glycoproteins through metabolic incorporation of unnatural sugars followed by click-reaction with organic fluorescent dyes. This approach allows for efficient labeling of diverse viral fusion glycoproteins on the surface of HIV pseudoviruses. Incorporation of a content marker into surface-labeled viral particles enables sensitive detection of single virus fusion with live cells. PMID:27033181

  6. Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery

    PubMed Central

    Burnham-Marusich, Amanda R.; Snodgrass, Casey J.; Johnson, Anna M.; Kiyoshi, Conrad M.; Buzby, Sarah E.; Gruner, Matt R.; Berninsone, Patricia M.

    2012-01-01

    Glycobiology research with Caenorhabditis elegans (C. elegans) has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family with established glycosylation in other species (actin). Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7). Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries. PMID:23152843

  7. Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.

    PubMed

    Moriya, Koko; Kimoto, Mayumi; Matsuzaki, Kanako; Kiwado, Aya; Takamitsu, Emi; Utsumi, Toshihiko

    2016-10-15

    To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus. PMID:27480498

  8. Measuring and modeling C flux rates through the central metabolic pathways in microbial communities using position-specific 13C-labeled tracers

    NASA Astrophysics Data System (ADS)

    Dijkstra, P.; van Groenigen, K.; Hagerty, S.; Salpas, E.; Fairbanks, D. E.; Hungate, B. A.; KOCH, G. W.; Schwartz, E.

    2012-12-01

    The production of energy and metabolic precursors occurs in well-known processes such as glycolysis and Krebs cycle. We use position-specific 13C-labeled metabolic tracers, combined with models of microbial metabolic organization, to analyze the response of microbial community energy production, biosynthesis, and C use efficiency (CUE) in soils, decomposing litter, and aquatic communities. The method consists of adding position-specific 13C -labeled metabolic tracers to parallel soil incubations, in this case 1-13C and 2,3-13C pyruvate and 1-13C and U-13C glucose. The measurement of CO2 released from the labeled tracers is used to calculate the C flux rates through the various metabolic pathways. A simplified metabolic model consisting of 23 reactions is solved using results of the metabolic tracer experiments and assumptions of microbial precursor demand. This new method enables direct estimation of fundamental aspects of microbial energy production, CUE, and soil organic matter formation in relatively undisturbed microbial communities. We will present results showing the range of metabolic patterns observed in these communities and discuss results from testing metabolic models.

  9. Metabolism of 35S- and 14C-labeled propylthiouracil in a model in vitro system containing thyroid peroxidase.

    PubMed

    Taurog, A; Dorris, M L; Guziec, F S; Uetrecht, J P

    1989-06-01

    In previous communications we described an in vitro model system containing highly purified thyroid peroxidase (TPO) for studying the mechanism of inhibition of thyroid hormone biosynthesis by the antithyroid drugs, 6-propylthiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI). We showed that inhibition of iodination of thyroglobulin in this system may be reversible or irreversible depending on the relative concentrations of iodide and drug and the TPO concentration. Metabolism of the drugs occurred under both conditions, but was more limited under irreversible conditions of inhibition. It was of interest to examine the nature of the drug metabolites associated with reversible and irreversible conditions of inhibition. For this purpose we have employed the 35S- and 14C-labeled drugs and a recently developed reverse phase HPLC procedure. Results of a similar study with MMI were reported in an earlier communication. In the present study we report our findings with PTU. Under conditions of reversible inhibition, PTU was readily metabolized and by 15 min was reduced to a few percent of the starting value. The earliest detectable metabolite with both [35S]- and [14C]PTU was the disulfide, which reached a peak in about 15 min and then slowly declined. Coincident with the decline in the disulfide was the appearance of more polar metabolites. In the case of [35S]PTU, these corresponded to sulfate/sulfite, PTU sulfonate, and a product tentatively identified as PTU sulfinate. The latter two were also observed as 14C-labeled metabolites produced from [14C]PTU. Two nonpolar desulfurated 14C-labeled metabolites were also observed. Surprisingly, these did not correspond to either propyluracil or propyldeoxyuracil, the anticipated most likely products of PTU desulfuration. The identity of these desulfurated metabolites of PTU in the TPO model system remains to be determined. Under conditions of irreversible inhibition of iodination, a relatively small fraction of PTU was

  10. Metabolic labeling of cellular glycoproteins with glucosamine: potential for erroneous interpretations due to nonenzymatic radiolabeling of proteins

    SciTech Connect

    Briles, E.I.B.; Updyke, T.V.

    1986-05-01

    Proteins, including serum proteins of culture media, become nonenzymatically radiolabeled under conditions used for metabolic labeling of cultured cells with glucosamine. This occurs even under sterile conditions in the absence of cells. Various commercial lots of /sup 3/H or /sup 14/C glcN gave similar results: approx. 0.7% of total label was incorporated into 20% serum (14 mg/ml protein) in 48 h at 37/sup 0/C. By SDS-PAGE fluorography, labeled serum bands correspond to Coomassie stained bands. Incorporation is linear with protein concentration and label input, shows biphasic kinetics (initial rapid rate within first 3 hr, followed by slower linear rate with no sign of saturation through 120 hr), and is temperature-dependent (no reaction at 0/sup 0/C; incorporation at 20/sup 0/C is approx. 45% of that at 37/sup 0/C). Poly-D-lysine is a better acceptor than protein: 0.5 mg/ml PL accepts as much label as 7 mg/ml protein. Incorporation is inhibited by excess unlabeled glcN and ethanolamine, but not by man, gal or glucose. However, when proteins were incubated with 160 mM glcN, SDS-PAGE bands were yellow-brown, suggesting the occurrence of Maillard-type reactions. Although the chemical mechanism(s) responsible for nonmetabolic radiolabeling by glcN are not clear at this point, the fact that it occurs represents a serious artifact which may lead to erroneous interpretation of data.

  11. New flaxseed orbitides: Detection, sequencing, and (15)N incorporation.

    PubMed

    Okinyo-Owiti, Denis P; Young, Lester; Burnett, Peta-Gaye G; Reaney, Martin J T

    2014-03-01

    Three new orbitides (cyclolinopeptides 17, 18, and 19) were identified in flaxseed (Linum usitatissimum L.) extracts without any form of purification. Their structures were elucidated by a combination of (15) N-labeling experiments and extensive tandem mass spectrometry (MS/MS) with electrospray ionization (ESI). Putative linear peptide sequences of the new orbitides were used as the query in the Basic Local Alignment Search Tool (BLAST) searches of flax genome database. These searches returned linear sequences for the putative precursors of cyclolinopeptides 17 and 19 among others. Cyclolinopeptide 18 contains MetO (O) and is not directly encoded, but is a product of post-translation modification of the Met present in 17. The identification of precursor proteins in flax mRNA transcripts and DNA sequences confirmed the occurrence and amino acid sequences of these orbitides as [1-9-NαC]-MLKPFFFWI, [1-9-NαC]-OLKPFFFWI, and [1-9-NαC]-GIPPFWLTL for cyclolinopeptides 17, 18, and 19, respectively. PMID:24408479

  12. Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing.

    PubMed

    Meissen, John K; Pirman, David A; Wan, Min; Miller, Emily; Jatkar, Aditi; Miller, Russell; Steenwyk, Rick C; Blatnik, Matthew

    2016-09-01

    Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture. PMID:27343766

  13. Carbon Metabolism of Soil microorganisms at Low Temperatures: Position-Specific 13C Labeled Glucose Reveals the Story

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Bore, E. K.; Halicki, S.; Kuzyakov, Y.; Dippold, M.

    2015-12-01

    Metabolic pathway activities at low temperature are not well understood, despite the fact that the processes are relevant for many soils globally and seasonally. To analyze soil metabolism at low temperature, isotopomeres of position-specifically 13C labeled glucose were applied at three temperature levels; +5, -5 -20 oC. In additon, one sterilization treatment with sodium azide at +5 oC was also performed. Soils were incubated for 1, 3 and 10 days while soil samples at -20 oC were additionally sampled after 30 days. The 13C from individual molecule position in respired CO2 was quantifed. Incorporation of 13C in bulk soil, extractable microbial biomass by chloroform fumigation extraction (CFE) and cell membranes of different microbial communities classified by 13C phospholipid fatty acid analysis (PLFA) was carried out. Our 13CO2 data showed a dominance of C-1 respiration at +5 °C for treatments with and without sodium azide, but total respiration for sodium azide inhibited treatments increased by 14%. In contrast, at -5 and -20 oC metabolic behavior showed intermingling of preferential respiration of the glucose C-4 and C-1 positions. Therefore, at +5 °C, pentose phosphate pathway activity is a dominant metabolic pathway used by microorganisms to metabolize glucose. The respiration increase due to NaN3 inhibition was attributed to endoenzymes released from dead organisms that are stabilized at the soil matrix and have access to suitable substrate and co-factors to permit their funtions. Our PLFA analysis showed that incorporation of glucose 13C was higher in Gram negative bacteria than other microbial groups as they are most competitive for LMWOS. Only a limited amount of microbial groups maintained their glucose utilizing activity at -5 and -20 °C and they strongly shifted towards a metabolization of glucose via both glycolysis and pentose phosphate pathways indicating both growth and cellular maintenance. This study revealed a remarkable microbial acitivity

  14. Nitrogen metabolism in Lignifying Pinus taeda cell cultures

    NASA Technical Reports Server (NTRS)

    van Heerden, P. S.; Towers, G. H.; Lewis, N. G.

    1996-01-01

    The primary metabolic fate of phyenylalanine, following its deamination in plants, is conscription of its carbon skeleton for lignin, suberin, flavonoid, and related metabolite formation. Since this accounts for approximately 30-40% of all organic carbon, an effective means of recycling the liberated ammonium ion must be operative. In order to establish how this occurs, the uptake and metabolism of various 15N-labeled precursors (15N-Phe, 15NH4Cl, 15N-Gln, and 15N-Glu) in lignifying Pinus taeda cell cultures was investigated, using a combination of high performance liquid chromatography, 15N NMR, and gas chromatograph-mass spectrometry analyses. It was found that the ammonium ion released during active phenylpropanoid metabolism was not made available for general amino acid/protein synthesis. Rather it was rapidly recycled back to regenerate phenylalanine, thereby providing an effective means of maintaining active phenylpropanoid metabolism with no additional nitrogen requirement. These results strongly suggest that, in lignifying cells, ammonium ion reassimilation is tightly compartmentalized.

  15. In vivo imaging and tracking of host-microbiota interactions via metabolic labeling of gut anaerobic bacteria.

    PubMed

    Geva-Zatorsky, Naama; Alvarez, David; Hudak, Jason E; Reading, Nicola C; Erturk-Hasdemir, Deniz; Dasgupta, Suryasarathi; von Andrian, Ulrich H; Kasper, Dennis L

    2015-09-01

    The intestine is densely populated by anaerobic commensal bacteria. These microorganisms shape immune system development, but understanding of host-commensal interactions is hampered by a lack of tools for studying the anaerobic intestinal environment. We applied metabolic oligosaccharide engineering and bioorthogonal click chemistry to label various commensal anaerobes, including Bacteroides fragilis, a common and immunologically important commensal. We studied the dissemination of B. fragilis after acute peritonitis and characterized the interactions of the intact microbe and its polysaccharide components in myeloid and B cell lineages. We were able to assess the distribution and colonization of labeled B. fragilis along the intestine, as well as niche competition after coadministration of multiple species of the microbiota. We also fluorescently labeled nine additional commensals (eight anaerobic and one microaerophilic) from three phyla common in the gut--Bacteroidetes, Firmicutes and Proteobacteria--as well as one aerobic pathogen (Staphylococcus aureus). This strategy permits visualization of the anaerobic microbial niche by various methods, including intravital two-photon microscopy and non-invasive whole-body imaging, and can be used to study microbial colonization and host-microbe interactions in real time. PMID:26280120

  16. In vivo imaging and tracking of host-microbiota interactions via metabolic labeling of gut anaerobic bacteria

    PubMed Central

    Geva-Zatorsky, Naama; Alvarez, David; Hudak, Jason E.; Reading, Nicola C.; Erturk-Hasdemir, Deniz; Dasgupta, Suryasarathi; von Andrian, Ulrich H.; Kasper, Dennis L.

    2015-01-01

    The intestine is densely populated by anaerobic commensal bacteria. These microorganisms shape immune system development, but our understanding of host–commensal interactions is hampered by a lack of tools for studying the anaerobic intestinal environment. We applied metabolic oligosaccharide engineering and bioorthogonal click-chemistry to label various commensal anaerobes, including Bacteroides fragilis, a common and immunologically important commensal. We studied the dissemination of B. fragilis following acute peritonitis, and characterized the interactions of the intact microbe and its polysaccharide components in myeloid and B cell lineages. The distribution and colonization of labeled B. fragilis along the intestine can be assessed, as well as niche competition following coadministration of multiple species of the microbiota. Nine additional anaerobic commensals (both gram-negative and gram-positive) from three phyla common in the gut—Bacteroidetes, Firmicutes, and Proteobacteria—and five families and one aerobic pathogen (Staphylococcus aureus) were also fluorescently labeled. This strategy permits visualization of the anaerobic microbial niche by various methods, including intravital two-photon microscopy and non-invasive whole-body imaging, and an approach to study microbial colonization and host–microbe interactions in real-time. PMID:26280120

  17. 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.

  18. Polyamine flux analysis by determination of heavy isotope incorporation from 13C, 15N-enriched amino acids into polyamines by LC-MS/MS.

    PubMed

    Cerrada-Gimenez, Marc; Häkkinen, Merja R; Vepsäläinen, Jouko; Auriola, Seppo; Alhonen, Leena; Keinänen, Tuomo A

    2012-02-01

    The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the (13)C, (15)N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC-MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine-N (1)-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC-MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux. PMID:21818565

  19. Site-Selective Synthesis of (15)N- and (13)C-Enriched Flavin Mononucleotide Coenzyme Isotopologues.

    PubMed

    Neti, Syam Sundar; Poulter, C Dale

    2016-06-17

    Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with (15)N and (13)C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis. PMID:27176708

  20. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling.

    PubMed

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-05-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a (13)CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1 The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and (13)C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions. PMID:26889011

  1. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling

    PubMed Central

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-01-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a 13CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1. The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and 13C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions. PMID:26889011

  2. Synthesis of 7-15N-Oroidin and Evaluation of Utility for Biosynthetic Studies of Pyrrole-Imidazole Alkaloids by Microscale1H-15N HSQC and FTMS†

    PubMed Central

    Wang, Yong-Gang; Morinaka, Brandon I.; Reyes, Jeremy Chris P.; Wolff, Jeremy H.; Romo, Daniel; Molinski, Tadeusz F.

    2010-01-01

    Numerous marine-derived pyrrole-imidazole alkaloids (PIAs), ostensibly derived from the simple precursor oroidin, 1a, have been reported and have garnered intense synthetic interest due to their complex structures and in some cases biological activity; however very little is known regarding their biosynthesis. We describe a concise synthesis of 7-15N-oroidin (1d) from urocanic acid and a direct method for measurement of 15N incorporation by pulse labeling and analysis by 1D 1H-15N HSQC NMR and FTMS. Using a mock pulse labeling experiment, we estimate the limit of detection (LOD) for incorporation of newly biosynthesized PIA by 1D 1H-15N HSQC to be 0.96 μg equivalent of 15N oroidin (2.4 nmole) in a background of 1500 μg unlabeled oroidin (about 1 part per 1600). 7-15N-Oroidin will find utility in biosynthetic feeding experiments with live sponges to provide direct information to clarify the pathways leading to more complex pyrrole-imidazole alkaloids. PMID:20095632

  3. Metabolic pathway for propionate utilization by phosphorus-accumulating organisms in activated sludge: 13C labeling and in vivo nuclear magnetic resonance.

    PubMed

    Lemos, Paulo C; Serafim, Luísa S; Santos, Margarida M; Reis, Maria A M; Santos, Helena

    2003-01-01

    In vivo 13C and 31P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-13C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV5, 59%; HV4, 5.0%; HV3, 1.1%; HV2, 3.5%; and HV1, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV4 is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism. PMID:12514001

  4. Retrieving nitrogen isotopic signatures from fresh leaf reflectance spectra: disentangling δ15N from biochemical and structural leaf properties

    PubMed Central

    Hellmann, Christine; Große-Stoltenberg, André; Lauströ, Verena; Oldeland, Jens; Werner, Christiane

    2015-01-01

    Linking remote sensing methodology to stable isotope ecology provides a promising approach to study ecological processes from small to large spatial scales. Here, we show that δ15N can be detected in fresh leaf reflectance spectra of field samples along a spatial gradient of increasing nitrogen input from an N2-fixing invasive species. However, in field data it is unclear whether δ15N directly influences leaf reflectance spectra or if the relationship is based on covariation between δ15N and foliar nitrogen content or other leaf properties. Using a 15N-labeling approach, we experimentally varied δ15N independently of any other leaf properties in three plant species across different leaf developmental and physiological states. δ15N could successfully be modeled by means of partial least squares (PLSs) regressions, using leaf reflectance spectra as predictor variables. PLS models explained 53–73% of the variation in δ15N within species. Several wavelength regions important for predicting δ15N were consistent across species and could furthermore be related to known absorption features of N-containing molecular bonds. By eliminating covariation with other leaf properties as an explanation for the relationship between reflectance and δ15N, our results demonstrate that 15N itself has an inherent effect on leaf reflectance spectra. Thus, our study substantiates the use of spectroscopic measurements to retrieve isotopic signatures for ecological studies and encourages future development. Furthermore, our results highlight the great potential of optical measurements for up-scaling isotope ecology to larger spatial scales. PMID:25983740

  5. New untargeted metabolic profiling combining mass spectrometry and isotopic labeling: application on Aspergillus fumigatus grown on wheat.

    PubMed

    Cano, Patricia M; Jamin, Emilien L; Tadrist, Souria; Bourdaud'hui, Pascal; Péan, Michel; Debrauwer, Laurent; Oswald, Isabelle P; Delaforge, Marcel; Puel, Olivier

    2013-09-01

    Characterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus , an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MS(n)) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC-HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins. PMID:23901908

  6. Box-modeling of 15N/14N in mammals.

    PubMed

    Balter, Vincent; Simon, Laurent; Fouillet, Hélène; Lécuyer, Christophe

    2006-03-01

    The 15N/14N signature of animal proteins is now commonly used to understand their physiology and quantify the flows of nutrient in trophic webs. These studies assume that animals are predictably 15N-enriched relative to their food, but the isotopic mechanism which accounts for this enrichment remains unknown. We developed a box model of the nitrogen isotope cycle in mammals in order to predict the 15N/14N ratios of body reservoirs as a function of time, N intake and body mass. Results of modeling show that a combination of kinetic isotope fractionation during the N transfer between amines and equilibrium fractionation related to the reversible conversion of N-amine into ammonia is required to account for the well-established approximately 4 per thousand 15N-enrichment of body proteins relative to the diet. This isotopic enrichment observed in proteins is due to the partial recycling of 15N-enriched urea and the urinary excretion of a fraction of the strongly 15N-depleted ammonia reservoir. For a given body mass and diet delta15N, the isotopic compositions are mainly controlled by the N intake. Increase of the urea turnover combined with a decrease of the N intake lead to calculate a delta15N increase of the proteins, in agreement with the observed increase of collagen delta15N of herbivorous animals with aridity. We further show that the low delta15N collagen values of cave bears cannot be attributed to the dormancy periods as it is commonly thought, but inversely to the hyperphagia behavior. This model highlights the need for experimental investigations performed with large mammals in order to improve our understanding of natural variations of delta15N collagen. PMID:16328553

  7. Metabolism of guanido-labeled (C-14)arginine in rats, mice, and man

    SciTech Connect

    Frondoza, C.G.; Trivedi, S.M.; Humphrey, R.L.; Goble, J.C.

    1980-01-01

    (6-/sup 14/C)arginine, injected intraperitoneally into normal rats, was cleared from the plasma with biphasic decay kinetics. Urinary excretion was efficient (32% of the 25-..mu..Ci dose within the first 24 h) with no preferential tissue retention. In mice, the effective duration of the radiotracer's availability for protein biosynthesis was less than 30 min. When the tracer was administered iv to patients with multiple myeloma, it was similarly cleared from the plasma with biphasic kinetics, and was excreted rapidly in the urine (22% of the dose within the first 24 h). In patients, the guanido-tagged arginine labeled only tumor M component, and the labeling was most intense in patients who had far advanced disease. Estimated radiation dose to humans from a 100-..mu..Ci injection was 10 mrads. These studies demonstrate the feasibility of in vivo labeling with (6-/sup 14/C)arginine, with minimal radiation hazard, thus providing a simple, sensitive, and specific method for monitoring the synthesis of the plasmacytoma M component in patients with multiple myeloma.

  8. Yeast membrane proteomics using leucine metabolic labelling: Bioinformatic data processing and exemplary application to the ER-intramembrane protease Ypf1.

    PubMed

    Nilse, Lars; Avci, Dönem; Heisterkamp, Patrick; Serang, Oliver; Lemberg, Marius K; Schilling, Oliver

    2016-10-01

    We describe in detail the usage of leucine metabolic labelling in yeast in order to monitor quantitative proteome alterations, e.g. upon removal of a protease. Since laboratory yeast strains are typically leucine auxotroph, metabolic labelling with trideuterated leucine (d3-leucine) is a straightforward, cost-effective, and ubiquitously applicable strategy for quantitative proteomic studies, similar to the widely used arginine/lysine metabolic labelling method for mammalian cells. We showcase the usage of advanced peptide quantification using the FeatureFinderMultiplex algorithm (part of the OpenMS software package) for robust and reliable quantification. Furthermore, we present an OpenMS bioinformatics data analysis workflow that combines accurate quantification with high proteome coverage. In order to enable visualization, peptide-mapping, and sharing of quantitative proteomic data, especially for membrane-spanning and cell-surface proteins, we further developed the web-application Proteator (http://proteator.appspot.com). Due to its simplicity and robustness, we expect metabolic leucine labelling in yeast to be of great interest to the research community. As an exemplary application, we show the identification of the copper transporter Ctr1 as a putative substrate of the ER-intramembrane protease Ypf1 by yeast membrane proteomics using d3-leucine isotopic labelling. PMID:27426920

  9. Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.

    PubMed

    Bindschedler, Laurence V; Mills, Davinia J S; Cramer, Rainer

    2012-01-01

    Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills. PMID:22665301

  10. Pharmacokinetics, metabolism, and excretion of (14)C-labeled belinostat in patients with recurrent or progressive malignancies.

    PubMed

    Calvo, Emiliano; Reddy, Guru; Boni, Valentina; García-Cañamaque, Lina; Song, Tao; Tjornelund, Jette; Choi, Mi Rim; Allen, Lee F

    2016-04-01

    Background Belinostat, a potent pan-inhibitor of histone deacetylase (HDAC) enzymes, is approved in the United States (US) for relapsed/refractory peripheral T-cell lymphoma. In nonclinical studies, bile and feces were identified as the predominant elimination routes (50-70 %), with renal excretion accounting for ~30-50 %. A Phase 1 human mass balance study was conducted to identify species-dependent variations in belinostat metabolism and elimination. Methods Patients received a single 30-min intravenous (IV) infusion of (14)C-labeled belinostat (1500 mg). Venous blood samples and pooled urine and fecal samples were evaluated using liquid chromatography - tandem mass spectroscopy for belinostat and metabolite concentrations pre-infusion through 7 days post-infusion. Total radioactivity was determined using liquid scintillation counting. Continued treatment with nonradiolabled belinostat (1000 mg/m(2) on Days 1-5 every 21 days) was permitted. Results Belinostat was extensively metabolized and mostly cleared from plasma within 8 h (N = 6), indicating that metabolism is the primary route of elimination. Systemic exposure for the 5 major metabolites was >20 % of parent, with belinostat glucuronide the predominant metabolite. Mean recovery of radioactive belinostat was 94.5 % ± 4.0 %, with the majority excreted within 48 and 96 h in urine and feces, respectively. Renal elimination was the principal excretion route (mean 84.8 % ± 9.8 % of total dose); fecal excretion accounted for 9.7 % ± 6.5 %. Belinostat was well tolerated, with mostly mild to moderate adverse events and no treatment-related severe/serious events. Conclusion Mass balance was achieved (~95 % mean recovery), with metabolism identified as the primary route of elimination. Radioactivity was predominantly excreted renally as belinostat metabolites. PMID:26769244

  11. Studies of metabolic pathways of trimebutine by simultaneous administration of trimebutine and its deuterium-labeled metabolite.

    PubMed

    Miura, Y; Chishima, S; Takeyama, S

    1989-01-01

    Trimebutine maleate (I), (+-)-2-dimethylamino-2-phenylbutyl 3,4,5-trimethoxybenzoate hydrogen maleate, and a deuterium-labeled sample of its hydrolyzed metabolite, 2-dimethylamino-2-phenylbutanol-d3 (II-d3), were simultaneously administered to experimental animals at an oral dose of 10 or 50 mumol/kg, and distribution ratios of the two alternative initial metabolic steps, i.e., ester hydrolysis and N-demethylation, were estimated by determining the composition of the urinary alcohol-moiety metabolites, II, and its mono- and di-demethylated metabolites, III and IV, by GC/MS. In dogs, the order of quantities of the metabolites from II-d3 was II much greater than III much greater than IV, showing predominance of conjugation over N-demethylation. However, this order was reversed when the amounts of the metabolites from I were compared, indicating that I was preferentially metabolized by N-demethylation followed by ester hydrolysis and conjugation in this order. In rats, a considerable proportion of I was presumed to be metabolized by ester hydrolysis before N-demethylation. In in vitro experiments employing the liver microsomes and homogenates of liver and small intestine from rats and dogs, it was found that both ester-hydrolizing and N-demethylating activities were higher in rats than in dogs, and the conjugating activity was higher in dogs than in rats. It was also found that I, having a high lipophilicity, was more susceptible to N-demethylation than less lipophilic II. These results from the in vitro experiments could account for the species differences in the distribution ratio of the metabolic pathways of I in vivo. PMID:2571489

  12. 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.

  13. High-throughput quantitation of metabolically labeled anionic glycoconjugates by scintillation proximity assay utilizing binding to cationic dyes.

    PubMed

    Rees-Milton, Karen J; Anastassiades, Tassos P

    2006-01-01

    Rapid, quantitative methods suited to a large number of samples are required for studies into the determination of disease etiology and in the evaluation of drugs and biological agents. This chapter describes an assay for anionic glycoconjugates (GCs), including glycosaminoglycans, which are major gene products of chondrocytes appearing in the extracellular matrix. The assay utilizes the electrostatic interaction between negatively charged sulfate and carboxyl groups of anionic GCs synthesized and secreted by chondrocytes with the cationic dye Alcian blue, immobilized to scintillant-coated 96-well plates. Metabolic labeling with D-[1, 6-3H (N)]-glucosamine allows all anionic GCs, including cartilage-specific and hyperglycosylated variants of fibronectin, to be quantitated. If Na235SO4 is used for the metabolic labeling instead, only glycosaminoglycans and proteoglycans will be quantitated. The samples are counted using a multi-detector instrument for scintillation proximity assays, such as the Wallac 1450 Microbeta Trilux, designed for detection of samples in 96-well plates and, as such, can be a high-throughput system. The bound anionic GCs can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after quantitation by elution with denaturing buffers. The method can be modified to include predigestion of the sample with a specific lyase, e.g., chondroitinase ABC or testicular hyaluronidase. To separate polyanions from other digested material after ethanol precipitation, the sample can be assayed as described in this chapter for a particular subtype of anionic GC. This assay addresses the need for high-throughput applications in arthritis and other medical and biological problems. PMID:17072016

  14. In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy.

    PubMed

    Xie, Ran; Dong, Lu; Du, Yifei; Zhu, Yuntao; Hua, Rui; Zhang, Chen; Chen, Xing

    2016-05-10

    Mammalian brains are highly enriched with sialoglycans, which have been implicated in brain development and disease progression. However, in vivo labeling and visualization of sialoglycans in the mouse brain remain a challenge because of the blood-brain barrier. Here we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for shuttling 9-azido sialic acid (9AzSia), a sialic acid reporter, into the brain to metabolically label sialoglycoconjugates, including sialylated glycoproteins and glycolipids. Subsequent bioorthogonal conjugation of the incorporated 9AzSia with fluorescent probes via click chemistry enabled fluorescence imaging of brain sialoglycans in living animals and in brain sections. Newly synthesized sialoglycans were found to widely distribute on neuronal cell surfaces, in particular at synaptic sites. Furthermore, large-scale proteomic profiling identified 140 brain sialylated glycoproteins, including a wealth of synapse-associated proteins. Finally, by performing a pulse-chase experiment, we showed that dynamic sialylation is spatially regulated, and that turnover of sialoglycans in the hippocampus is significantly slower than that in other brain regions. The LABOR strategy provides a means to directly visualize and monitor the sialoglycan biosynthesis in the mouse brain and will facilitate elucidating the functional role of brain sialylation. PMID:27125855

  15. De Novo Proteome Analysis of Genetically Modified Tumor Cells By a Metabolic Labeling/Azide-alkyne Cycloaddition Approach*

    PubMed Central

    Ballikaya, Seda; Lee, Jennifer; Warnken, Uwe; Schnölzer, Martina; Gebert, Johannes; Kopitz, Jürgen

    2014-01-01

    Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome. PMID:25225355

  16. Metabolism of /sup 14/C-labeled doxylamine succinate (Bendectin) in the rhesus monkey (Macaca mulatta)

    SciTech Connect

    Slikker, W. Jr.; Holder, C.L.; Lipe, G.W.; Korfmacher, W.A.; Thompson, H.C. Jr.; Bailey, J.R.

    1986-05-01

    The time-course of the metabolic fate of (/sup 14/C)doxylamine was determined after the p.o. administration of 13 mg/kg doxylamine succinate as Bendectin plus (/sup 14/C)doxylamine succinate to the rhesus monkey. Urine and plasma samples were analyzed by reversed-phase high performance liquid chromatography (HPLC), chemical derivatization, and mass spectrometry. The cumulative 48-hr urinary metabolic profile contained 81% of the administered radiolabeled dose and consisted of at least six radiolabeled peaks. They were peak 1: unknown polar metabolites (8% of dose); peak 2: 2-(1-phenyl-1-(2-pyridinyl)ethoxy) acetic acid, 1-(1-phenyl-1(2-pyridinyl)ethoxy) methanol, and another minor metabolite(s) (31%); peak 3: doxylamine-N-oxide (1%); peak 4a: N,N-didesmethyldoxylamine (17%); peak 4b: doxylamine (4%); and peak 5: N-desmethyldoxylamine (20%). The plasma metabolic profile was the same as the urinary profile except for the absence of doxylamine-N-oxide. The maximum plasma concentrations and elapsed time to attain these concentrations were as follows. Peak 1: 540 ng/mL, 4 hr; peak 2: 1700 ng/mL, 1 hr; peak 4a: 430 ng/mL, 4 hr; peak 4b: 930 ng/mL, 2 hr; and peak 5: 790 ng/mL, 2 hr. These data suggest that in the monkey, doxylamine metabolism follows at least four pathways: a minor pathway to the N-oxide; a minor pathway to unknown polar metabolites; a major pathway to mono- and didesmethyldoxylamine via successive N-demethylation; and a major pathway to side-chain cleavage products (peak 2) via direct side-chain oxidation and/or deamination.

  17. 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.

  18. Stable Isotope Labeled Tracers for Metabolic Pathway Elucidation by GC-MS and FT-MS

    PubMed Central

    Higashi, Richard M.; Fan, Teresa W-M.; Lorkiewicz, Pawel K.; Moseley, Hunter N.B.; Lane, Andrew N.

    2015-01-01

    Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics is poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, Stable Isotope Resolved Metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks, in a wide range of experimental systems, including human subjects. MS offers a wide range of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS affordable by many individual laboratories, to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter will focus on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM. PMID:25270929

  19. Backbone and sidechain 1H, 15N and 13C assignments of the KSR1 CA1 domain

    PubMed Central

    Koveal, Dorothy; Pinheiro, Anderson S.; Peti, Wolfgang; Page, Rebecca

    2014-01-01

    The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [13C, 15N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain. PMID:20737253

  20. Effective Estimation of Dynamic Metabolic Fluxes Using 13C Labeling and Piecewise Affine Approximation: From Theory to Practical Applicability

    PubMed Central

    Schumacher, Robin; Wahl, S. Aljoscha

    2015-01-01

    The design of microbial production processes relies on rational choices for metabolic engineering of the production host and the process conditions. These require a systematic and quantitative understanding of cellular regulation. Therefore, a novel method for dynamic flux identification using quantitative metabolomics and 13C labeling to identify piecewise-affine (PWA) flux functions has been described recently. Obtaining flux estimates nevertheless still required frequent manual reinitalization to obtain a good reproduction of the experimental data and, moreover, did not optimize on all observables simultaneously (metabolites and isotopomer concentrations). In our contribution we focus on measures to achieve faster and robust dynamic flux estimation which leads to a high dimensional parameter estimation problem. Specifically, we address the following challenges within the PWA problem formulation: (1) Fast selection of sufficient domains for the PWA flux functions, (2) Control of over-fitting in the concentration space using shape-prescriptive modeling and (3) robust and efficient implementation of the parameter estimation using the hybrid implicit filtering algorithm. With the improvements we significantly speed up the convergence by efficiently exploiting that the optimization problem is partly linear. This allows application to larger-scale metabolic networks and demonstrates that the proposed approach is not purely theoretical, but also applicable in practice. PMID:26690237

  1. Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling

    PubMed Central

    Hu, Fanghao; Wei, Lu; Zheng, Chaogu; Shen, Yihui

    2014-01-01

    Choline is a small molecule that occupies a key position in the biochemistry of all living organisms. Recent studies have strongly implicated choline metabolites in cancer, atherosclerosis and nervous system development. To detect choline and its metabolites, existing physical methods such as magnetic resonance spectroscopy and positron emission tomography, are often limited by the poor spatial resolution and substantial radiation dose. Fluorescence imaging, although with submicrometer resolution, requires introduction of bulky fluorophores and thus is difficult in labeling the small choline molecule. By combining the emerging bond-selective stimulated Raman scattering microscopy with metabolic incorporation of deuterated choline, herein we have achieved high resolution imaging of choline-containing metabolites in living mammalian cell lines, primary hippocampal neurons and multicellular organism C. elegans. Different subcellular distributions of choline metabolites are observed between cancer cells and non-cancer cells, which may reveal functional difference in the choline metabolism and lipid-mediated signaling events. In neurons, choline incorporation is visualized within both soma and neurites, where choline metabolites are more evenly distributed compared to the protein. Furthermore, choline localization is also observed in the pharynx region of C. elegans larvae, consistent with its organogenesis mechanism. These applications demonstrate the potential of isotope-based stimulated Raman scattering microscopy for future choline-related disease detection and development monitoring in vivo. PMID:24555181

  2. Noninvasive imaging of intracellular lipid metabolism in macrophages by Raman microscopy in combination with stable isotopic labeling.

    PubMed

    Matthäus, Christian; Krafft, Christoph; Dietzek, Benjamin; Brehm, Bernhard R; Lorkowski, Stefan; Popp, Jürgen

    2012-10-16

    Monocyte-derived macrophages play a key role in atherogenesis because their transformation into foam cells is responsible for deposition of lipids in plaques within arterial walls. The appearance of cytosolic lipid droplets is a hallmark of macrophage foam cell formation, and the molecular basics involved in this process are not well understood. Of particular interest is the intracellular fate of different individual lipid species, such as fatty acids or cholesterol. Here, we utilize Raman microscopy to image the metabolism of such lipids and to trace their subsequent storage patterns. The combination of microscopic information with Raman spectroscopy provides a powerful molecular imaging method, which allows visualization at the diffraction limit of the employed laser light and biochemical characterization through associated spectral information. In order to distinguish the molecules of interest from other naturally occurring lipids spectroscopically, deuterium labels were introduced. Intracellular distribution and metabolic changes were observed for serum albumin-complexed palmitic and oleic acid and cholesterol and quantitatively evaluated by monitoring the increase in CD scattering intensities at 0.5, 1, 3, 6, 24, 30, and 36 h. This approach may also allow for investigating the cellular trafficking of other molecules, such as nutrients, metabolites, and drugs. PMID:22954250

  3. Nutrient depletion and metabolic profiles in breast carcinoma cell lines measured with a label-free platform.

    PubMed

    Demmel, F; Brischwein, M; Wolf, P; Huber, F; Pfister, C; Wolf, B

    2015-07-01

    The response of two well-characterized human breast cancer cell lines (MCF-7 and MDA-MB-231) to a series of nutrient deficiencies is investigated with a label-free cell assay platform. The motivation of the research is to analyze adaptive responses of tumor cell metabolism and to find limiting conditions for cell survival. The platform measures extracellular values of pH and dissolved oxygen saturation to provide data of extracellular acidification rates and oxygen uptake rates. Additional electric cell substrate impedance sensing and bright-field cell imaging supports the data interpretation by providing information about cell morphological parameters. A sequential administration of nutrient depletions does not cause metabolic reprogramming, since the ratios of oxygen uptake to acidification return to their basal values. While the extracellular acidification drops sharply upon reduction of glucose and glutamine, the oxygen uptake is not affected. In contrast to other published data, cell death is not observed when both glucose and glutamine are depleted and cell proliferation is not inhibited, at least in MCF-7 cultures. It is assumed that residual concentrations of nutrients from the serum component are able to maintain cell viability when delivered regularly by active flow like in the cell assay platform, and, in a similar way, under physiological conditions. PMID:26015442

  4. Positron emission reconstruction tomography for the assessment of regional myocardial metabolism by the administration of substrates labeled with cyclotron produced radionuclides

    NASA Technical Reports Server (NTRS)

    Ter-Pogossian, M. M.; Hoffman, E. J.; Weiss, E. S.; Coleman, R. E.; Phelps, M. E.; Welch, M. J.; Sobel, B. E.

    1975-01-01

    A positron emission transverse tomograph device was developed which provides transaxial sectional images of the distribution of positron-emitting radionuclides in the heart. The images provide a quantitative three-dimensional map of the distribution of activity unencumbered by the superimposition of activity originating from regions overlying and underlying the plane of interest. PETT is used primarily with the cyclotron-produced radionuclides oxygen-15, nitrogen-13 and carbon-11. Because of the participation of these atoms in metabolism, they can be used to label metabolic substrates and intermediary molecules incorporated in myocardial metabolism.

  5. Measurement of Histone Methylation Dynamics by One-Carbon Metabolic Isotope Labeling and High-energy Collisional Dissociation Methylation Signature Ion Detection

    PubMed Central

    Tang, Hui; Tian, Bing; Brasier, Allan R.; Sowers, Lawrence C.; Zhang, Kangling

    2016-01-01

    Accumulating evidence suggests that cellular metabolites and nutrition levels control epigenetic modifications, including histone methylation. However, it is not currently possible to measure the metabolic control of histone methylation. Here we report a novel detection method to monitor methyl transfer from serine to histones through the one-carbon metabolic pathway, using stable-isotope labeling and detection of lysine methylation signature ions generated in high-energy-dissociation (HCD) tandem mass spectrometry. This method is a long-needed tool to study the metabolic control of histone methylation. PMID:27530234

  6. Metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate.

    PubMed

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-01-01

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling, while LRRK2 is implicated in the pathogenesis of Parkinson's disease. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with (32)P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing (32)P-orthophosphate. The (32)P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography ((32)P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. PMID:24084685

  7. Absorption, distribution, metabolism, and excretion of [14C]-labeled naloxegol in healthy subjects

    PubMed Central

    Bui, Khanh; She, Fahua; Hutchison, Michael; Brunnström, Åsa; Sostek, Mark

    2015-01-01

    Objective: To characterize the absorption, distribution, metabolism, and excretion of naloxegol, a PEGylated derivative of the µ-opioid antagonist naloxone, in healthy male subjects. Materials and methods: [14C]-Labeled naloxegol (27 mg, 3.43 MBq) was administered as an oral solution to 6 fasted subjects. Blood, fecal, and urine samples were collected predose and at various intervals postdose. Naloxegol and its metabolites were quantified or identified by liquid chromatography with radiometric or mass spectrometric detection. Pharmacokinetic parameters were calculated for each subject, and metabolite identification was performed by liquid chromatography with parallel radioactivity measurement and mass spectrometry. Results: Naloxegol was rapidly absorbed, with a maximum plasma concentration (geometric mean) of 51 ng/mL reached before 2 hours after dosing. A second peak in the observed naloxegol and [14C] plasma concentration-time profiles was observed at ~ 3 hours and was likely due to enterohepatic recycling of parent naloxegol. Distribution to red blood cells was negligible. Metabolism of [14C]-naloxegol was rapid and extensive and occurred via demethylation and oxidation, dealkylation, and shortening of the polyethylene glycol chain. Mean cumulative recovery of radioactivity was 84.2% of the total dose, with ~ 68.9% recovered within 96 hours of dosing. Fecal excretion was the predominant route of elimination, with mean recoveries of total radioactivity in feces and urine of 67.7% and 16.0%, respectively. Unchanged naloxegol accounted for ~ 1/4 of the radioactivity recovered in feces. Conclusions: Naloxegol was rapidly absorbed and cleared via metabolism, with predominantly fecal excretion of parent and metabolites. PMID:26329350

  8. Mammalian DNA δ15N exhibits 40‰ intramolecular variation and is unresponsive to dietary protein level

    PubMed Central

    Strable, Maggie S.; Tschanz, Carolyn L.; Varamini, Behzad; Chikaraishi, Yoshito; Ohkouchi, Naohiko; Brenna, J. Thomas

    2014-01-01

    We report the first high precision characterization of molecular and intramolecular δ15N of nucleosides derived from mammalian DNA. The influence of dietary protein level on brain amino acids and deoxyribonucleosides was determined to investigate whether high protein turnover would alter amino acid 15N or 13C. Pregnant guinea pig dams were fed control diets, or high or low levels of dietary protein throughout gestation, and all pups were fed control diets. Cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ15N and δ13C characterized. Mean diet δ15N = 0.45±0.33‰, compared to cerebellar whole tissue and DNA δ15N = +4.1±0.7‰ and −4.5±0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ15N were +1.4±0.4, −2.1±0.9, −7.2±0.3, and −10.4±0.5‰, respectively. There were no changes in amino acid or deoxyribonucleoside δ15N due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ15N originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ15NASP-PUR, δ15NASP-PYR, δ15NGLN, and δ15NGLY +11.9±2.3‰, +7.0±2.0‰, −9.1±2.4‰, and −31.8±8.9‰, respectively. A subset of twelve amino acids from food and brain had mean δ15N of 4.3±3.2‰ and 13.8±3.1‰, respectively, and δ15N for gly and asp were 12.6±2.2‰ and 15.2±0.8‰, respectively. A separate isotope tracer study detected no significant turnover of cerebellar DNA in the first six months of life. The large negative δ15N difference between gly and cerebellar purine N at the gly (7) position implies either that there is a major isotope effect during DNA synthesis, or that in utero gly has a different isotope ratio during rapid growth and metabolism than in adult life. Our data show that cerebellar nucleoside intramolecular δ15N vary over more than

  9. LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.

    PubMed

    Frey, Alexander J; Feldman, Daniel R; Trefely, Sophie; Worth, Andrew J; Basu, Sankha S; Snyder, Nathaniel W

    2016-05-01

    Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters. PMID:26968563

  10. Effect of Photoperiod on the Metabolism of Deuterium-Labeled Gibberellin A53 in Spinach 1

    PubMed Central

    Gianfagna, Thomas; Zeevaart, Jan A. D.; Lusk, William J.

    1983-01-01

    Application of gibberellin A53 (GA53) to short-day (SD)-grown spinach (Spinacia oleracea L.) plants caused an increase in petiole length and leaf angle similar to that found in plants transferred to long days (LD). [2H] GA53 was fed to plants in SD, LD, and in a SD to LD transition experiment, and the metabolites were identified by gas chromatography with selected ion monitoring. After 2, 4, or 6 SD, [2H]GA53 was converted to [2H]GA19 and [2H]GA44. No other metabolites were detected. After 2 LD, only [2H] GA20 was identified. In the transition experiment in which plants were given 4 SD followed by 2 LD, all three metabolites were found. The results demonstrate unequivocally that GA19, GA20, and GA44 are metabolic products of GA53, and strongly suggest that photoperiod regulates GA metabolism, in part, by controlling the conversion of GA19 to GA20. PMID:16662988

  11. 1H, 13C and 15N resonance assignments of URNdesign, a computationally redesigned RRM protein

    SciTech Connect

    Dobson, Neil; Dantas, Gautam; Varani, Gabriele

    2005-10-01

    Protein design represents one of the great challenges of computational structural biology. The ability to successfully design new proteins would allow us to generate new reagents and enzymes, while at the same time providing us with an understanding of the principles of protein stability. Here we report 1H, 15N and 13C resonance assignments of a redesigned U1A protein, URNdesign. U1A has been studied extensively by our group and hence was chosen as a design target. For the assignments we sued 2D and 3D heteronuclearNMR experiments with uniformly 13C, 15N-labeled URNdesign. The assignments for the backbone NH, CO,Ca and Cb nuclei are 94%complete. Sidechain 1Hand13C, aromatic andQ/NNH2 resonances are essentially complete with guanidinium and K NH3 residues unassigned. BMRB deposit with accession number 6493

  12. Comparative metabolism of 14C-labeled apixaban in mice, rats, rabbits, dogs, and humans.

    PubMed

    Zhang, Donglu; He, Kan; Raghavan, Nirmala; Wang, Lifei; Mitroka, James; Maxwell, Brad D; Knabb, Robert M; Frost, Charles; Schuster, Alan; Hao, Feng; Gu, Zheming; Humphreys, W Griffith; Grossman, Scott J

    2009-08-01

    The metabolism and disposition of [(14)C]apixaban, a potent, reversible, and direct inhibitor of coagulation factor Xa, were investigated in mice, rats, rabbits, dogs, and humans after a single oral administration and in incubations with hepatocytes. In plasma, the parent compound was the major circulating component in mice, rats, dogs, and humans. O-Demethyl apixaban sulfate (M1) represented approximately 25% of the parent area under the time curve in human plasma. This sulfate metabolite was present, but in lower amounts relative to the parent, in plasma from mice, rats, and dogs. Rabbits showed a plasma metabolite profile distinct from that of other species with apixaban as a minor component and M2 (O-demethyl apixaban) and M14 (O-demethyl apixaban glucuronide) as prominent components. The fecal route was a major elimination pathway, accounting for >54% of the dose in animals and >46% in humans. The urinary route accounted for <15% of the dose in animals and 25 to 28% in humans. Apixaban was the major component in feces of every species and in urine of all species except rabbit. M1 and M2 were common prominent metabolites in urine and feces of all species as well as in bile of rats and humans. In vivo metabolite profiles showed quantitative differences between species and from in vitro metabolite profiles, but all human metabolites were found in animal species. After intravenous administration of [(14)C]apixaban to bile duct-cannulated rats, the significant portion (approximately 22%) of the dose was recovered as parent drug in the feces, suggesting direct excretion of the drug from gastrointestinal tracts of rats. Overall, apixaban was effectively eliminated via multiple elimination pathways in animals and humans, including oxidative metabolism, and direct renal and intestinal excretion. PMID:19420130

  13. 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

  14. Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

    PubMed Central

    Starkey, Jonathan M.; Zhao, Yingxin; Sadygov, Rovshan G.; Haidacher, Sigmund J.; LeJeune, Wanda S.; Dey, Nilay; Luxon, Bruce A.; Kane, Maureen A.; Napoli, Joseph L.; Denner, Larry; Tilton, Ronald G.

    2010-01-01

    Background Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes. Methodology/Principal Findings Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, 18O- and 16O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change ≥1.5 and p≤0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARβ/δ mRNA. Conclusions/Significance Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in

  15. Acetylene inhibition of N2O reduction in laboratory soil and groundwater denitrification assays: evaluation by 15N tracer and 15N site preference of N2O

    NASA Astrophysics Data System (ADS)

    Weymann, Daniel; Well, Reinhard; Lewicka-Szczebak, Dominika; Lena, Rohe

    2013-04-01

    The measurement of denitrification in soils and aquifers is still challenging and often enough associated with considerable experimental effort and high costs. Against this background, the acetylene inhibition technique (AIT) applied in laboratory soil and groundwater denitrification assays is by far the most effective approach. However, this method has been largely criticized, as it is susceptible to underestimate denitrification rates and adds an additional carbon source to the substrates to be investigated. Here we provide evidence that the AIT is not necessarily an inappropriate approach to measure denitrification, that its reliability depends on the drivers governing the process, and that the 15N site preference of N2O (SP) may serve as a tool to assess this reliability. Two laboratory batch experiments were conducted, where sandy aquifer material and a peat soil were incubated as slurries. We established (i) a standard anaerobic treatment by adding KNO3 (10 mg N L-1), (ii) an oxygen treatment by adding KNO3 and O2 (5 mg L-1), and (iii) a glucose treatment by adding KNO3 supplemented with glucose (200 mg C L-1). Both experiments were run under 10 % (v/v) acetylene atmosphere and as 15N tracer treatments using labeled K15NO3 (60 atom % 15N). In the case of the standard anaerobic treatments, we found a very good agreement of denitrification potential obtained by the AIT and 15N tracer methods. SP of N2O of the AIT samples from this treatment ranged between -4.8 and 2.6 ‰ which is indicative for N2O production during bacterial denitrification but not for N2O reduction to N2. In contrast, we observed substantial underestimation of denitrification by AIT for the glucose treatments compared to the 15N method, i.e. denitrification was underestimated by 36 % (sandy aquifer material) and 47 % (peat soil). SP of N2O of the AIT samples from this treatment ranged between 4.5 and 9.6 ‰, which suggests occurrence of bacterial N2O reduction. In the case of the oxygen

  16. Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

    NASA Astrophysics Data System (ADS)

    Smeekens, Johanna M.; Chen, Weixuan; Wu, Ronghu

    2015-04-01

    Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide- N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

  17. Validation of the doubly-labeled water (H/sup 3/H/sup 18/O) method for measuring water flux and energy metabolism in tenebrionid beetles

    SciTech Connect

    Cooper, P.D.

    1981-01-01

    Doubly-labeled water (H/sup 3/H/sup 18/O) has been used to determine water flux and energy metabolism in a variety of vertebrates. This study examines the applicability of this technique to arthropods. The theory of the technique depends upon the assumption that doubly-labeled water introduced into the animal's body water equilibrates with water and carbon dioxide by the action of carbonic anhydrase. Tritium (/sup 3/H) is lost from the animal only with water while oxygen-18 is lost with both water and carbon dioxide. The difference bwtween the rates of loss of the two isotopes is proportional to CO/sub 2/ loss rate. Validation of the use of tritiated water for measuring water flux was accomplished by comparing gravimetric measurements of water gain with flux rates determined by loss of tritiated water. At room humidity, an overestimate for influx calculated from labeled water calculations was found, averaging 12 mg H/sub 2/O (g.d)/sup -1/. Comparison of CO/sub 2/ loss rate determined isotopically with rates of CO/sub 2/ loss determined by standard metabolic rates also yielded overestimates for the isotopic technique, overestimates ranging between 20 and 30%. The relevance of this for studies using labeled water for studying water fluxes and free metabolism of free-ranging arthropods is discussed.

  18. Safety and efficacy of polycalcium for improving biomarkers of bone metabolism: a 4-week open-label clinical study.

    PubMed

    Choi, Jae-Suk; Park, Mi-Yeon; Kim, Jong-Dae; Cho, Hyung Rae; Choi, In Soon; Kim, Joo-Wan

    2013-03-01

    Polycalcium is a mixture of Polycan and calcium lactate-gluconate 1:9 (w/w) with demonstrated antiosteoporosis activity in vitro and in vivo studies. These studies were a 4-week open-label, single-center trial to evaluate the efficacy of oral Polycalcium on bone metabolism and safety. In total, 30 healthy women (range 40-60 years) were administered 400 mg of Polycalcium for 4 weeks. The primary efficacy parameter was urinary deoxypyridinoline (DPYR) levels, and serum osteocalcin (OSC), bone-specific alkaline phosphatase (BALP), urinary cross-linked C-telopeptide of type-1 collagen (CTx), urinary cross-linked N-telopeptide of type-1 collagen (NTx), calcium (Ca), and phosphorus (P) levels, which were evaluated for comparison before and after administration of Polycalcium. After 4 weeks of Polycalcium administration, 27 subjects completed the test plan. Three subjects withdrew their consent to participate. The values of blood OSC, BALP, serum Ca, and serum P from baseline to 4 weeks of treatment were changed by -28.44%, 14.37%, 6.11%, and 1.42%, respectively. Biomarkers of bone resorption: urinary DPYR, serum CTx, serum NTx, urinary Ca, and urinary P, at baseline after 4 weeks of treatment were changed by -13.40%, 6.67%, -5.13%, -22.43%, and -3.04%, respectively. Additionally, when considering the subjects' adverse effects and the results of the blood and urine tests over the 4-week trial period, the dose of 400  mg Polycalcium showed efficacy for improving bone metabolism and was well tolerated and safe. Polycalcium was apparently safe and efficacious. PMID:23477624

  19. Safety and Efficacy of Polycalcium for Improving Biomarkers of Bone Metabolism: A 4-Week Open-Label Clinical Study

    PubMed Central

    Choi, Jae-Suk; Park, Mi-Yeon; Kim, Jong-Dae; Cho, Hyung Rae

    2013-01-01

    Abstract Polycalcium is a mixture of Polycan and calcium lactate–gluconate 1:9 (w/w) with demonstrated antiosteoporosis activity in vitro and in vivo studies. These studies were a 4-week open-label, single-center trial to evaluate the efficacy of oral Polycalcium on bone metabolism and safety. In total, 30 healthy women (range 40–60 years) were administered 400 mg of Polycalcium for 4 weeks. The primary efficacy parameter was urinary deoxypyridinoline (DPYR) levels, and serum osteocalcin (OSC), bone-specific alkaline phosphatase (BALP), urinary cross-linked C-telopeptide of type-1 collagen (CTx), urinary cross-linked N-telopeptide of type-1 collagen (NTx), calcium (Ca), and phosphorus (P) levels, which were evaluated for comparison before and after administration of Polycalcium. After 4 weeks of Polycalcium administration, 27 subjects completed the test plan. Three subjects withdrew their consent to participate. The values of blood OSC, BALP, serum Ca, and serum P from baseline to 4 weeks of treatment were changed by −28.44%, 14.37%, 6.11%, and 1.42%, respectively. Biomarkers of bone resorption: urinary DPYR, serum CTx, serum NTx, urinary Ca, and urinary P, at baseline after 4 weeks of treatment were changed by −13.40%, 6.67%, −5.13%, −22.43%, and −3.04%, respectively. Additionally, when considering the subjects' adverse effects and the results of the blood and urine tests over the 4-week trial period, the dose of 400 mg Polycalcium showed efficacy for improving bone metabolism and was well tolerated and safe. Polycalcium was apparently safe and efficacious. PMID:23477624

  20. 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

  1. 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

  2. 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.

  3. Study of the metabolism of /sup 13/C labeled substrates by /sup 13/C NMR spectroscopy of intact cells, tissues, and organs

    SciTech Connect

    Matwiyoff, N.A.; London, R.E.; Hutson, J.Y.

    1982-01-01

    Carbon-13 nuclear magnetic resonance spectroscopy, in conjunction with carbon-13 labeling, has become an important analytical technique for the study of biological systems and biologically important molecules. The growing list of its well established applications to isolated molecules in solution includes the investigation of: metabolic pathways; the microenvironments of ligands bound to proteins; the architecture and dynamics of macromolecules; the structures of coenzymes and other natural products; and the mechanisms of reactions. Recently interest has been reawakened in the use of the technique for the study of metabolic pathways and structural components in intact organelles, cells, and tissues. The promise and problems in the use of /sup 13/C labeling in such investigations can be illustrated by the results on suspensions of the yeast, Candida utilis.

  4. Automated microbial metabolism laboratory. [design of advanced labeled release experiment based on single addition of soil and multiple sequential additions of media into test chambers

    NASA Technical Reports Server (NTRS)

    1974-01-01

    The design and rationale of an advanced labeled release experiment based on single addition of soil and multiple sequential additions of media into each of four test chambers are outlined. The feasibility for multiple addition tests was established and various details of the methodology were studied. The four chamber battery of tests include: (1) determination of the effect of various atmospheric gases and selection of that gas which produces an optimum response; (2) determination of the effect of incubation temperature and selection of the optimum temperature for performing Martian biochemical tests; (3) sterile soil is dosed with a battery of C-14 labeled substrates and subjected to experimental temperature range; and (4) determination of the possible inhibitory effects of water on Martian organisms is performed initially by dosing with 0.01 ml and 0.5 ml of medium, respectively. A series of specifically labeled substrates are then added to obtain patterns in metabolic 14CO2 (C-14)O2 evolution.

  5. Isotopologue profiling of the listerial N-metabolism.

    PubMed

    Kutzner, Erika; Kern, Tanja; Felsl, Angela; Eisenreich, Wolfgang; Fuchs, Thilo M

    2016-04-01

    The nitrogen (N-) sources and the relative contribution of a nitrogenous nutrient to the N-pool of the gram-positive pathogen Listeria monocytogenes are largely unknown. Therefore, (15) N-isotopologue profiling was established to study the N-metabolism of L. monocytogenes. The pathogen was grown in a defined minimal medium supplemented with potential (15) N-labeled nutrients. The bacteria were harvested and hydrolysed under acidic conditions, and the resulting amino acids were analysed by GC-MS, revealing (15) N-enrichments and isotopomeric compositions of amino acids. The differential (15) N-profiles showed the substantial and simultaneous usage of ammonium, glutamine, methionine, and, to a lower extent, the branched-chain amino acids valine, leucine, and isoleucine for anabolic purposes, with a significant preference for ammonium. In contrast, arginine, histidine and cysteine were directly incorporated into proteins. L. monocytogenes is able to replace glutamine with ethanolamine or glucosamine as amino donors for feeding the core N-metabolism. Perturbations of N-fluxes caused by gene deletions demonstrate the involvement of ethanolamine ammonia lyase, and suggest a role of the regulator GlnK of L. monocytogenes distinct from that of Escherichia coli. The metabolism of nitrogenous nutrients reflects the high flexibility of this pathogenic bacterium in exploiting N-sources that could also be relevant for its proliferation during infection. PMID:26699934

  6. An automated growth enclosure for metabolic labeling of Arabidopsis thaliana with 13C-carbon dioxide - an in vivo labeling system for proteomics and metabolomics research

    PubMed Central

    2011-01-01

    Background Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with 13C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate 13C from 13CO2 gas. Because of the high cost of 13CO2 it is critical that the design conserve the labeled gas. Results A fully enclosed automated plant growth enclosure has been designed and assembled where the system simultaneously monitors humidity, temperature, pressure and 13CO2 concentration with continuous adjustment of humidity, pressure and 13CO2 levels controlled by a computer running LabView software. The enclosure is mounted on a movable cart for mobility among growth environments. Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides. Conclusion The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS. Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs. PMID:21310072

  7. 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.

  8. Amine-reactive neutron-encoded labels for highly plexed proteomic quantitation.

    PubMed

    Hebert, Alexander S; Merrill, Anna E; Stefely, Jonathan A; Bailey, Derek J; Wenger, Craig D; Westphall, Michael S; Pagliarini, David J; Coon, Joshua J

    2013-11-01

    We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between (13)C and (15)N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference-relative to its nearest neighbor-so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration. PMID:23882030

  9. Amine-reactive Neutron-encoded Labels for Highly Plexed Proteomic Quantitation*

    PubMed Central

    Hebert, Alexander S.; Merrill, Anna E.; Stefely, Jonathan A.; Bailey, Derek J.; Wenger, Craig D.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.

    2013-01-01

    We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between 13C and 15N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference—relative to its nearest neighbor—so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration. PMID:23882030

  10. 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. PMID:25935409

  11. Natural (15)N Abundance in Key Amino Acids from Lamb Muscle: Exploring a New Horizon in Diet Authentication and Assessment of Feed Efficiency in Ruminants.

    PubMed

    Cantalapiedra-Hijar, Gonzalo; Ortigues-Marty, Isabelle; Schiphorst, Anne-Marie; Robins, Richard J; Tea, Illa; Prache, Sophie

    2016-05-25

    Natural (15)N abundance (δ(15)N) varies between individual amino acids (AAs). We hypothesized that δ(15)N of nontransaminating and essential AAs ("source" AAs, such as phenylalanine) present in animal tissues could be used as a marker of dietary origin, whereas δ(15)N of transaminating AAs ("trophic" AAs, such as glutamic acid) could give more detailed insights into animal feed efficiency. Two diets based on dehydrated Lucerne pellets were tested in growing lambs, which promoted different feed efficiencies. No dietary effects were noted on δ(15)N of any AAs analyzed in lamb muscle. In addition, δ(15)N of phenylalanine was unexpectedly similar to that of glutamic acid, suggesting that δ(15)N of AAs is significantly derived from the metabolism of the rumen microbiota and, thus, are not suited for diet authentication in ruminants. In contrast, the δ(15)N of transaminating AAs facilitates an improved prediction of animal feed efficiency compared to the classical isotopic bulk N analysis. PMID:27148901

  12. 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.

  13. Theoretical and experimental study of 15N NMR protonation shifts.

    PubMed

    Semenov, Valentin A; Samultsev, Dmitry O; Krivdin, Leonid B

    2015-06-01

    A combined theoretical and experimental study revealed that the nature of the upfield (shielding) protonation effect in 15N NMR originates in the change of the contribution of the sp(2)-hybridized nitrogen lone pair on protonation resulting in a marked shielding of nitrogen of about 100 ppm. On the contrary, for amine-type nitrogen, protonation of the nitrogen lone pair results in the deshielding protonation effect of about 25 ppm, so that the total deshielding protonation effect of about 10 ppm is due to the interplay of the contributions of adjacent natural bond orbitals. A versatile computational scheme for the calculation of 15N NMR chemical shifts of protonated nitrogen species and their neutral precursors is proposed at the density functional theory level taking into account solvent effects within the supermolecule solvation model. PMID:25891386

  14. Dark CO/sub 2/ fixation and amino acid metabolism in symbiotic N/sub 2/-fixing systems. Labeling studies with /sup 14/C and /sup 13/N-labeled tracers. [Roots of soybean plants and alders

    SciTech Connect

    Coker, G.T. III

    1982-01-01

    Amino acid metabolism was examined by monitoring the amino acids labeled with (/sup 14/C) incorporated during dark CO/sub 2/ fixation and with (/sup 13/N) incorporated from /sup 13/NH/sub 4/+, /sup 13/NO/sub 3/- or (/sup 13/N)N/sub 2/. Label from /sup 14/CO/sub 2/ was directly incorporated in soybean roots and the N/sub 2/-fixing root nodules of soybeans and alders. The products of dark CO/sub 2/ fixation were primarily amino and organic acids. The distribution of label incorporated from /sup 14/CO/sub 2/ into amino acids depended on the plant species and the nitrigen source. The major labeled amino acids in roots and nodules of soybean plants dependent on N/sub 2/ were aspartate and glutamate; in alder nodules, citrulline; in roots of soybean plants treated with NO/sub 3/-, asparagine; and in roots of soybean plants treated with NH/sub 4/+, asparagine and glutamine. Asparagine was the major amino acid transported out of the soybean root system. Experiments indicated that asparagine was synthesized directly from aspartate. After exposure to /sup 14/CO/sub 2/, the specific activity of glutamine was consistently higher than that of glutamate in soybean nodules and roots of plants treated with NO/sub 3/-. This was taken as evidence that there were two pools of glutamate, only one of which was associated with glutamine synthesis. Alder and soybean nodules and roots were incubated with /sup 13/N-labeled tracers. Those tissues incubated with /sup 13/NH/sub 4/+ had a higher ratio of (/sup 13/N)glutamine to (/sup 13/N)glutamate that similar tissues exposed to /sup 13/NO/sub 3/- or (/sup 13/N)N/sub 2/. An explanation for these results based on the relative rates of glutamine and glutamate synthesis is discussed.

  15. Metabolic fate of the (14)C-labeled herbicide clodinafop-propargyl in a sediment-water system.

    PubMed

    Yuan, Ye; Weitzel, Pascal; Schäffer, Andreas; Schmidt, Burkhard

    2015-01-01

    The metabolic fate of (14)C-phenyl-labeled herbicide clodinafop-propargyl ((14)C-CfP) was studied for 28 days in lab assays using a sediment-water system derived from a German location. Mineralization was 5.21% of applied (14)C after 28 days exhibiting a distinct lag phase until day 14 of incubation. Portions of radioactivity remaining in water phases decreased at moderate rate to 18.48% after 28 days; 62.46% were still detected in water after 14 days. Soxhlet extraction of the sediment using acetonitrile released 35.56% of applied (14)C with day 28, while 33.99% remained as non-extractable residues. A remarkable increase of bound (14)C was observed between 14 and 28 days correlating with the distinct increase of mineralization. No correlation was found throughout incubation with microbial activity of the sediment as determined by dimethyl sulfoxide reduction. Dissolved oxygen and pH value of water phases remained almost constant for 28 days. Analyses of Soxhlet extracts of the sediment and ethyl acetate extracts of water phases by radio-TLC and radio-HPLC revealed that CfP was rapidly cleaved to free acid clodinafop (Cf), which was further (bio-) transformed. DT50 values (based on radio-HPLC) were below 1 day (CfP) and slightly above 28 days (Cf). Further metabolites were not detected. Fractionation of humic and non-humic components of the sediment demonstrated that CfP's non-extractable residues were predominantly associated with fulvic acids up to 14 days of incubation (3.36%), whereas after 28 days, the majority of radioactivity was found in the humin/mineral fraction (13.30% of applied (14)C). Due to high-performance size-exclusion chromatography of the fulvic acids fraction derived from assays incubated for 28 days, this portion of (14)C was firmly, possibly covalently bound to fulvic acids and did not consist of CfP or Cf. Using an isolation strategy comprising preincubation of sediment with CfP and mineralization of (14)C-CfP as criterion, a microorganism

  16. 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. PMID:24746715

  17. 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. PMID:26470275

  18. Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N.

    PubMed Central

    Nissim, Itzhak; Horyn, Oksana; Luhovyy, Bohdan; Lazarow, Adam; Daikhin, Yevgeny; Nissim, Ilana; Yudkoff, Marc

    2003-01-01

    The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate. PMID:12935293

  19. Impacts of proline on the central metabolism of an industrial erythromycin-producing strain Saccharopolyspora erythraea via (13)C labeling experiments.

    PubMed

    Hong, Ming; Huang, Mingzhi; Chu, Ju; Zhuang, Yingping; Zhang, Siliang

    2016-08-10

    Saccharopolyspora erythraea E3 is an important industrial strain for erythromycin production and knowledge on its metabolism is limited. In the present work, (13)C labeling experiments were conducted to characterize the metabolism of S. erythraea E3. We found that S. erythraea E3 was difficult to grow on minimal medium with glucose as sole carbon source and the addition of proline remarkably improved the cell growth. The activity of EMP pathway was very low and ED pathway was alternatively the main glucose utilization pathway. The addition of proline resulted in remarkable changes in the fluxes of central metabolism. The fluxes in PP pathway, in TCA cycle and in ED pathway were 90% higher, 64% and 31% lower on Glc/Pro than on Glc, respectively. The maintenance energy on Glc/Pro was 58.4% lower than that on Glc. The energy charge was lower on Glc than on Glc/Pro, indicating that the cells on Glc suffered from energy burden. This study elucidates the impacts of proline on the central metabolism of S. erythraea and deepens the understanding of its metabolism. PMID:27215341

  20. Metabolism

    MedlinePlus

    Metabolism refers to all the physical and chemical processes in the body that convert or use energy, ... Tortora GJ, Derrickson BH. Metabolism. In: Tortora GJ, Derrickson BH. Principles of Anatomy and Physiology . 14th ed. Hoboken, NJ: John H Wiley and Sons; 2013: ...

  1. 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

  2. Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy-D-manno-oct-2-ulosonic acid.

    PubMed

    Dumont, Marie; Lehner, Arnaud; Vauzeilles, Boris; Malassis, Julien; Marchant, Alan; Smyth, Kevin; Linclau, Bruno; Baron, Aurélie; Mas Pons, Jordi; Anderson, Charles T; Schapman, Damien; Galas, Ludovic; Mollet, Jean-Claude; Lerouge, Patrice

    2016-02-01

    In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth. PMID:26676799

  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. Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor.

    PubMed Central

    Rouquette-Jazdanian, Alexandre K; Pelassy, Claudette; Breittmayer, Jean-Philippe; Cousin, Jean-Louis; Aussel, Claude

    2002-01-01

    Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts. PMID:11964165

  5. 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

  6. 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.

  7. Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

    PubMed Central

    Snyder, Nathaniel W.; Tombline, Gregory; Worth, Andrew J.; Parry, Robert C.; Silvers, Jacob A.; Gillespie, Kevin P.; Basu, Sankha S.; Millen, Jonathan; Goldfarb, David S.; Blair, Ian A.

    2015-01-01

    Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants. PMID:25572876

  8. PARTITIONING THE RELATIVE INFLUENCE OF SOIL N, MYCORRHIZAE, AND FOLIAR N UPTAKE ON FOLIAR δ15N PATTERNS: CAN WE DETECT FOLIAR UPTAKE OF REACTIVE N?

    NASA Astrophysics Data System (ADS)

    Vallano, D.; Sparks, J. P.

    2009-12-01

    Vegetation is an important sink for atmospheric reactive N in N-limited systems and may be capable of incorporating reactive N compounds directly into leaves through the foliar uptake pathway. A proxy for atmospheric reactive N entering vegetation would be useful to estimate the impact of direct foliar N uptake on plant metabolism. Natural abundance foliar N isotopic composition (δ15N) is a practical tool for this purpose because plant-available N sources often have different isotopic compositions. Current understanding of foliar δ15N suggests these values primarily represent the integration of soil δ15N, direct foliar N uptake, mycorrhizal fractionation, and within-plant fractionations. Using a potted plant mesocosm system, we estimated the influence of mycorrhizae on foliar δ15N patterns in red maple (Acer rubrum) seedlings along an N deposition gradient in New York State. We found that mycorrhizal associations altered foliar δ15N in red maple seedlings from 0.03 - 1.01‰ across sites. Along the same temporal and spatial scales, we examined the influence of soil δ15N, foliar N uptake, and mycorrhizae on foliar δ15N in adult stands of American beech (Fagus grandifolia), black birch (Betula lenta), red maple (A. rubrum), and red oak (Quercus rubra). Using multiple regression models, atmospheric NO2 concentration explained 0%, 69%, 23%, and 45% of the residual variation in foliar δ15N remaining in American beech, red maple, red oak, and black birch, respectively, after accounting for soil δ15N. Our results suggest that foliar δ15N may be used to estimate pollution-derived atmospheric reactive N entering vegetation via the foliar N uptake pathway.

  9. Increasing plant use of organic nitrogen with elevation is reflected in nitrogen uptake rates and ecosystem delta15N.

    PubMed

    Averill, Colin; Finzi, Adrien

    2011-04-01

    It is hypothesized that decreasing mean annual temperature and rates of nitrogen (N) cycling causes plants to switch from inorganic to organic forms of N as the primary mode of N nutrition. To test this hypothesis, we conducted field experiments and collected natural-abundance delta15N signatures of foliage, soils, and ectomycorrhizal sporocarps along a steep elevation-climate gradient in the White Mountains, New Hampshire, USA. Here we show that with increasing elevation organic forms of N became the dominant source of N taken up by hardwood and coniferous tree species based on dual-labeled glycine uptake analysis, an important confirmation of an emerging theory for the biogeochemistry of the N cycle. Variation in natural abundance foliar delta15N with elevation was also consistent with increasing organic N uptake, though a simple, mass balance model demonstrated that the uptake of delta15N depleted inorganic N, rather than fractionation upon transfer of N from mycorrhizal fungi, best explains variations in foliar delta15N with elevation. PMID:21661551

  10. Norlittorine and norhyoscyamine identified as products of littorine and hyoscyamine metabolism by (13)C-labeling in Datura innoxia hairy roots.

    PubMed

    Al Balkhi, Mohamad Houssam; Schiltz, Séverine; Lesur, David; Lanoue, Arnaud; Wadouachi, Anne; Boitel-Conti, Michèle

    2012-02-01

    The presence of two compounds, norlittorine and norhyoscyamine, has been reported in leaves and roots of Datura innoxia; however their metabolic origin in the tropane alkaloid pathway has remained unknown. Precise knowledge of this pathway is a necessary pre-requisite to optimize the production of hyoscyamine and scopolamine in D. innoxia hairy root cultures. The exact structure of norlittorine and norhyoscyamine was confirmed by LC-MS/MS and NMR analyses. Isotopic labeling experiments, using [1-(13)C]-phenylalanine, [1'-(13)C]-littorine and [1'-(13)C]-hyoscyamine, combined with elicitor treatments, using methyl jasmonate, coronalon and 1-aminocyclopropane-1-carboxylic acid, were used to investigate the metabolic origin of the N-demethylated tropane alkaloids. The results suggest that norlittorine and norhyoscyamine are induced under stress conditions by conversion of littorine and hyoscyamine. We propose the N-demethylation of tropane alkaloids as a mechanism to detoxify cells in overproducing conditions. PMID:22083085

  11. Structure and Metabolic-Flow Analysis of Molecular Complexity in a (13) C-Labeled Tree by 2D and 3D NMR.

    PubMed

    Komatsu, Takanori; Ohishi, Risa; Shino, Amiu; Kikuchi, Jun

    2016-05-10

    Improved signal identification for biological small molecules (BSMs) in a mixture was demonstrated by using multidimensional NMR on samples from (13) C-enriched Rhododendron japonicum (59.5 atom%) cultivated in air containing (13) C-labeled carbon dioxide for 14 weeks. The resonance assignment of 386 carbon atoms and 380 hydrogen atoms in the mixture was achieved. 42 BSMs, including eight that were unlisted in the spectral databases, were identified. Comparisons between the experimental values and the (13) C chemical shift values calculated by density functional theory supported the identifications of unlisted BSMs. Tracing the (13) C/(12) C ratio by multidimensional NMR spectra revealed faster and slower turnover ratios of BSMs involved in central metabolism and those categorized as secondary metabolites, respectively. The identification of BSMs and subsequent flow analysis provided insight into the metabolic systems of the plant. PMID:27060701

  12. Multimodality imaging can predict the metabolic response of unresectable colorectal liver metastases to radioembolization therapy with Yttrium-90 labeled resin microspheres

    NASA Astrophysics Data System (ADS)

    Flamen, Patrick; Vanderlinden, Bruno; Delatte, Philippe; Ghanem, Ghanem; Ameye, Lieveke; Van Den Eynde, Marc; Hendlisz, Alain

    2008-11-01

    Selective internal radiotherapy (SIRT) using Yttrium-90 labeled resin microspheres is increasingly used for the radioembolization of unresectable liver metastases of colorectal cancer (CRC). The treatment can be simulated by scintigraphy with Tc99m-labeled macroaggregates of albumin (MAA). The aim of the study was to develop a predictive dosimetric model for SIRT and to validate it by correlating results with the metabolic treatment response. The simulation of the dosimetry was performed by mathematically converting all liver voxel MAA-SPECT uptake values to the absolute Y90 activity. The voxel values were then converted to a simulated absorbed dose (Gy) using simple MIRD formalism. The metabolic response was defined as the change in total lesion glycolysis (TLG) on FDG-PET. A total of 39 metastatic liver lesions were studied in eight evaluable patients. The mean administered Y90 activity was 1.69 GBq (range: 1.33-2.04 GBq). The median change in TLG of the lesions was 48%. The median (95% CI) simulated absorbed dose (Gy) was 20 Gy (1-68 Gy) and 46 Gy (22-110 Gy) in the poor (<50% TLG change) and the good responders (TLG change > 50%), respectively. Using a simple cut-off value of 1 for the MAA-tumor-to-normal uptake ratio, a significant metabolic response was predicted with a sensitivity of 89% (17/19), a specificity of 65% (13/20), a positive predictive value of 71% (17/24) and a negative predictive value of 87% (13/15). Integrated multimodality imaging allows prediction of metabolic response post radioembolization using Y90-resin microspheres, and should be used for patient selection.

  13. The vibrational spectra of [ 15N 2]-succinonitrile

    NASA Astrophysics Data System (ADS)

    Fengler, O. I.

    2001-07-01

    For the first time, the infrared and Raman spectra of [ 15N 2]-succinonitrile are presented and discussed in detail. Assignments of the vibrational bands of its two rotational conformers gauche and trans, respectively, have been made for both infrared and Raman spectra. The assignments were based on a recent ab-initio force field calculation for succinonitrile, taking into account the vibrational frequencies of other succinonitrile isotopomers. There are differences in the frequencies of the vibrational bands due to the mass increase in the cyanide groups, which have been analysed in depth.

  14. The vibrational spectra of [15N2]-succinonitrile.

    PubMed

    Fengler, O I

    2001-07-01

    For the first time, the infrared and Raman spectra of [15N2]-succinonitrile are presented and discussed in detail. Assignments of the vibrational bands of its two rotational conformers gauche and trans, respectively, have been made for both infrared and Raman spectra. The assignments were based on a recent ab-initio force field calculation for succinonitrile, taking into account the vibrational frequencies of other succinonitrile isotopomers. There are differences in the frequencies of the vibrational bands due to the mass increase in the cyanide groups, which have been analysed in depth. PMID:11471715

  15. 15N NMR chemical shifts in papaverine decomposition products

    NASA Astrophysics Data System (ADS)

    Czyrski, Andrzej; Girreser, Ulrich; Hermann, Tadeusz

    2013-03-01

    Papaverine can be easily oxidized to papaverinol, papaveraldine and 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1-a]isoquinolinium chloride. On addition of alkali solution the latter compound forms 2-(2-carboxy-4,5-dimethoxyphenyl)-6,7-dimethoxyisoquinolinium inner salt. Together with these structures the interesting 13-(3,4-dimethoxyphenyl)-2,3,8,9-tetramethoxy-6a-12a-diazadibenzo[a,g]fluorenylium chloride is discussed, which is formed in the Gadamer-Schulemann reaction of papaverine as a side product. This letter reports the 15N NMR spectra of the above mentioned compounds.

  16. Experimental plant for simultaneous production of (14)N and (15)N by (15)N/(14)N exchange in NO, NO(2)-HNO(3) system under pressure.

    PubMed

    Axente, Damian; Marcu, Cristina; Muresan, Ancuţa; Kaucsar, Martin; Misan, Ioan; Popeneciu, Gabriel; Gligan, Nicolae; Cristea, Gabriela

    2010-06-01

    An experimental study on (14)N and (15)N simultaneous separation using the chemical exchange in NO, NO(2)-HNO(3) system under pressure is presented. The influence of the pressure and of the interstage 10 M HNO(3) flow rate on the separation of (14)N and (15)N was measured on a packed column with product and waste refluxers. At steady state and 1.8 atm (absolute), the isotopic concentration at the bottom of the separation column was 0.563 at% (15)N, and in the top of the column was 0.159 at% (15)N. The height equivalent to a theoretical plate and interstage 10 M HNO(3) flow rate values, obtained in these experimental conditions, allows the separation of (14)N highly depleted of (15)N and of (15)N at 99 at% (15)N concentration. PMID:20582793

  17. Metabolic power of European starlings Sturnus vulgaris during flight in a wind tunnel, estimated from heat transfer modelling, doubly labelled water and mask respirometry.

    PubMed

    Ward, S; Möller, U; Rayner, J M V; Jackson, D M; Nachtigall, W; Speakman, J R

    2004-11-01

    It is technically demanding to measure the energetic cost of animal flight. Each of the previously available techniques has some disadvantage as well advantages. We compared measurements of the energetic cost of flight in a wind tunnel by four European starlings Sturnus vulgaris made using three independent techniques: heat transfer modelling, doubly labelled water (DLW) and mask respirometry. We based our heat transfer model on thermal images of the surface temperature of the birds and air flow past the body and wings calculated from wing beat kinematics. Metabolic power was not sensitive to uncertainty in the value of efficiency when estimated from heat transfer modelling. A change in the assumed value of whole animal efficiency from 0.19 to 0.07 (the range of estimates in previous studies) only altered metabolic power predicted from heat transfer modelling by 13%. The same change in the assumed value of efficiency would cause a 2.7-fold change in metabolic power if it were predicted from mechanical power. Metabolic power did not differ significantly between measurements made using the three techniques when we assumed an efficiency in the range 0.11-0.19, although the DLW results appeared to form a U-shaped power-speed curve while the heat transfer model and respirometry results increased linearly with speed. This is the first time that techniques for determining metabolic power have been compared using data from the same birds flying under the same conditions. Our data provide reassurance that all the techniques produce similar results and suggest that heat transfer modelling may be a useful method for estimating metabolic rate. PMID:15531650

  18. /sup 13/N-labeled L-amino acids for in vivo assessment of local myocardial metabolism

    SciTech Connect

    Baumgartner, F.J.; Barrio, J.R.; Henze, E.; Schelbert, H.R.; MacDonald, N.S.; Phelps, M.E.; Kuhl, D.E.

    1981-06-01

    The hot cell synthesis of sterile, pyrogen-free /sup 13/N-labeled L-amino acids was accomplished by employing the appropriate immobilized enzymes on a CNBr-activated Sepharose support and using remote, semiautomated systems. The syntheses were completed 6-12 min after cyclotron production of (/sup 13/N)ammonia. Myocardial time-activity curves after intracoronary injection of /sup 13/N-labeled L-amino acids in dogs were triexponential in both normal and ischemic myocardium. Higher retention of /sup 13/N activity was observed in ischemic segments. Positron computed tomography imaging also showed increased uptake of /sup 13/N-labeled L-glutamate and L-alanine in ischemic segments compared with normal myocardium when blood flow corrections were made. Myocardial transaminases are primarily responsible for the observed retention fractions. It suggests the participation of the carbon skeletons of these amino acids in the Krebs cycle.

  19. Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

    NASA Technical Reports Server (NTRS)

    Tunlid, A.; Odham, G.; Findlay, R. H.; White, D. C.

    1985-01-01

    Sensitive detection of cellular components from specific groups of microbes can be utilized as 'signatures' in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacterial) cell wall, can be detected reproducibly. Enrichments of D-[15N]alanine determined in E. coli grown with [15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M - HF)- and (M - F or M + H - HF)- formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10(3)-10(4) cells, as a function of the 15N-14N ratio.

  20. Development of a Microfluidic-Based Optical Sensing Device for Label-Free Detection of Circulating Tumor Cells (CTCs) Through Their Lactic Acid Metabolism

    PubMed Central

    Chiu, Tzu-Keng; Lei, Kin-Fong; Hsieh, Chia-Hsun; Hsiao, Hung-Bo; Wang, Hung-Ming; Wu, Min-Hsien

    2015-01-01

    This study reports a microfluidic-based optical sensing device for label-free detection of circulating tumor cells (CTCs), a rare cell species in blood circulation. Based on the metabolic features of cancer cells, live CTCs can be quantified indirectly through their lactic acid production. Compared with the conventional schemes for CTC detection, this label-free approach could prevent the biological bias due to the heterogeneity of the surface antigens on cancer cells. In this study, a microfluidic device was proposed to generate uniform water-in-oil cell-encapsulating micro-droplets, followed by the fluorescence-based optical detection of lactic acid produced within the micro-droplets. To test its feasibility to quantify cancer cells, experiments were carried out. Results showed that the detection signals were proportional to the number of cancer cells within the micro-droplets, whereas such signals were insensitive to the existence and number of leukocytes within. To further demonstrate its feasibility for cancer cell detection, the cancer cells with known cell number in a cell suspension was detected based on the method. Results revealed that there was no significant difference between the detected number and the real number of cancer cells. As a whole, the proposed method opens up a new route to detect live CTCs in a label-free manner. PMID:25808775

  1. 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

    Labeling plant material with heavy isotopes of carbon and nitrogen can produce a traceable nutrient signal that can be followed into the different trophic levels and decomposer food web. We treated 60 tree saplings with 13C-enriched CO2 gas and 15N-enriched ammonium nitrate over a three-month period to create dually-labeled plant material for future decomposition experiments. The trees included both early (Red maple, Sweetgum, Tulip poplar) and late (American beech, White oak) successional deciduous tree species, and a conifer, White pine. We constructed a 2.4 m × 2.4 m × 2.4 m environmental chamber that was climate-controlled using an air conditioning system. An Arduino microcontroller interfaced with a Vaisala GMP343 CO2 probe maintained a CO2 concentration between 500-520 ppm by controlling a solenoid valve on the CO2 tank regulator. The trees were placed into the chamber in August 2012 and remained until senescence unless they were lost to death or disease. Ammonium nitrate was added twice, in September and October. Leaf samples were collected prior to the start of the experiment and after senescence, whereas root samples were collected only in December. Samples were dried, ground and analyzed using an isotope ratio mass spectrometer. American beech and White oak had 40% mortality, and 34% of tulip poplar trees were removed because of powdery mildew overgrowth or death. Most tulip poplar trees exhibited a second leaf out following senescence in late September. Nearly 1 kg of litter was produced with tulip poplar representing over half of the total mass. Levels of enrichment varied greatly by species. Beech (-14.2‰) and White oak (-4.8‰) had low levels of enrichment in comparison to early successional species such as Sweetgum (41.7‰) and Tulip poplar (30.7‰ [first leaf fall] and 238.0‰ [second leaf fall]). Leaf enrichment with 15N followed a similar pattern, though it was achieved at a higher level with δ15N values varying from 271.6‰ to 1354.2

  2. Evidence for transketolase-like TKTL1 flux in CHO cells based on parallel labeling experiments and (13)C-metabolic flux analysis.

    PubMed

    Ahn, Woo Suk; Crown, Scott B; Antoniewicz, Maciek R

    2016-09-01

    The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in (13)C metabolic flux analysis ((13)C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-(13)C]glucose+[4,5,6-(13)C]glucose, [2-(13)C]glucose+[4,5,6-(13)C]glucose, and [3-(13)C]glucose+[4,5,6-(13)C]glucose and developed a new method to measure (13)C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells. PMID:27174718

  3. UPTAKE, METABOLISM, AND ELIMINATION OF 14C-LABELED 1,2,4-TRICHLOROBENZENE IN RAINBOW TROUT AND CARP

    EPA Science Inventory

    Fingerling rainbow trout (Salmo gairdneri) were exposed to 14C-labeled 1,2,4-trichlorobenzene (TCB) for 8 h in a static exposure (0.018 mg/1) or for 35 d in a continuous-flow exposure (0.020 mg/1) followed by a subsequent elimination period. For the 2 d after the 8-h exposure, th...

  4. Specific deuterium labelling and computerized gas chromatography -- mass spectrometry in studies on the metabolism in vivo of a steroid sulphate in the rat.

    PubMed

    Baillie, T A; Eriksson, H; Herz, J E; Sjövall, J

    1975-06-16

    The metabolism of 3beta-hydroxy-5alpha-pregnan-20-one sulphate was studied in bile fistula rats and in isolated perfused livers. Computerized gas chromatography--mass spectrometry, in combination with specific deuterium-labelling, was employed to follow the metabolic transformations. Male animals excreted metabolites into bile more rapidly than females, a finding which could be correlated with the preferential formation of glucuronide conjugates in the male liver. The major metabolic pathway in male rats involved the steps: hydrolysis, 2alpha-hydroxylation, oxidoreduction at C-3 and glucuronide conjugation, yielding 2alpha, 3alpha-dihydroxy-5alpha-pregnan-20-one glucuronide as the major metabolite. Only traces of the injected steroid sulphate were detected in bile from male animals. In contrast, the administered compound was the major steroid excreted in bile of female rats, where the main metabolite was identified as 3beta,15beta-dihydroxy-5alpha-pregnan-20-one sulphate. A minor metabolite, 3beta,16alpha-dihydroxy-5alpha-pregnan-20-one, was found as a monosulphate in female rats and as both a disulphate and a glucuronide conjugate in male rats. The deuterium content of the sulphated 15beta-and 16alpha-hydroxylated metabolites was consistent with metabolic pathways involving direct hydroxylation of the injected steroid sulphate. The results obtained from the liver perfusions were essentially the same as those from the experiments with bile fistula animals. This indicates that all the observed metabolic reactions took place in the liver. PMID:1175601

  5. Regional, seasonal and interspecific variation in 15N and 13C in sympatric mouse lemurs

    NASA Astrophysics Data System (ADS)

    Rakotondranary, S. Jacques; Struck, Ulrich; Knoblauch, Christian; Ganzhorn, Jörg U.

    2011-11-01

    Madagascar provides some of the rare examples where two or more primate species of the same genus and with seemingly identical niche requirements occur in sympatry. If congeneric primate species co-occur in other parts of the world, they differ in size in a way that is consistent with Hutchinson's rule for coexisting species, or they occupy different ecological niches. In some areas of Madagascar, mouse lemurs do not follow these "rules" and thus seem to violate one of the principles of community ecology. In order to understand the mechanisms that allow coexistence of sympatric congeneric species without obvious niche differentiation, we studied food composition of two identical sized omnivorous mouse lemur species, Microcebus griseorufus and M. murinus with the help of stable isotope analyses ( δ 15N and δ 13C). The two species are closely related sister species. During the rich season, when food seems abundant, the two species do not differ in their nitrogen isotope composition, indicating that the two species occupy the same trophic level. But they differ in their δ 13C values, indicating that M. griseorufus feeds more on C4 and CAM (Crassulacean-acid-metabolism) plants than M. murinus. During the lean season, M. murinus has lower δ 15N values, indicating that the two species feed at different trophic levels during times of food shortage. Hybrids between the two species showed intermediate food composition. The results reflect subtle differences in foraging or metabolic adaptations that are difficult to quantify by traditional observations but that represent possibilities to allow coexistence of species.

  6. Intraskeletal isotopic compositions (δ(13) C, δ(15) N) of bone collagen: nonpathological and pathological variation.

    PubMed

    Olsen, Karyn C; White, Christine D; Longstaffe, Fred J; von Heyking, Kristin; McGlynn, George; Grupe, Gisela; Rühli, Frank J

    2014-04-01

    Paleodiet research traditionally interprets differences in collagen isotopic compositions (δ(13) C, δ(15) N) as indicators of dietary distinction even though physiological processes likely play some role in creating variation. This research investigates the degree to which bone collagen δ(13) C and δ(15) N values normally vary within the skeleton and examines the influence of several diseases common to ancient populations on these isotopic compositions. The samples derive from two medieval German cemeteries and one Swiss reference collection and include examples of metabolic disease (rickets/osteomalacia), degenerative joint disease (osteoarthritis), trauma (fracture), infection (osteomyelitis), and inflammation (periostitis). A separate subset of visibly nonpathological skeletal elements from the German collections established normal intraindividual variation. For each disease type, tests compared bone lesion samples to those near and distant to the lesions sites. Results show that normal (nonpathological) skeletons exhibit limited intraskeletal variation in carbon- and nitrogen-isotope ratios, suggesting that sampling of distinct elements is appropriate for paleodiet studies. In contrast, individuals with osteomyelitis, healed fractures, and osteoarthritis exhibit significant intraskeletal differences in isotope values, depending on whether one is comparing lesions to near or to distant sites. Skeletons with periostitis result in significant intraskeletal differences in nitrogen isotope values only, while those with rickets/osteomalacia do not exhibit significant intraskeletal differences. Based on these results, we suggest that paleodiet researchers avoid sampling collagen at or close to lesion sites because the isotope values may be reflecting both altered metabolic processes and differences in diet relative to others in the population. PMID:24374993

  7. Nitrogen Dynamics in Stream Wood Samples Incubated with [14C]Lignocellulose and Potassium [15N]Nitrate †

    PubMed Central

    Aumen, Nicholas G.; Bottomley, Peter J.; Gregory, Stan V.

    1985-01-01

    Surface wood samples obtained from a Douglas fir log (Pseudotsuga menziesii) in a Pacific Northwest stream were incubated in vitro with [14C]lignocellulose in a defined mineral salts medium supplemented with 10 mg of N liter−1 of 15N-labeled NO3− (50 atom% 15N). Evolution of 14CO2, distribution and isotopic dilution of 15N, filtrate N concentrations, and the rates of denitrification, N2 fixation, and respiration were measured at 6, 12, and 18 days of incubation. The organic N content of the lignocellulose-wood sample mixture had increased from 132 μg of N to a maximum of 231 μg of N per treatment after 6 days of incubation. Rates of [14C]lignocellulose decomposition were greatest during the first 6 days and then began to decline over the remaining 12 days. Total CO2 evolution was also highest at day 6 and declined steadily over the remaining duration of the incubation. Filtrate NH4+-N increased from background levels to a final value of 57 μg of N per treatment. Filtrate NO3− N completely disappeared by day 6, and organic N showed a slight decline between days 12 and 18. The majority of the 15N that could be recovered appeared in the particulate organic fraction by day 6 (41 μg of N), and the filtrate NH4+ N fraction contained 11 μg of 15N by day 18. The 15N enrichment values of the filtrate NH4+ and the inorganic N associated with the particulate fraction had increased to approximately 20 atom% 15N by 18 days of incubation, whereas the particulate organic fraction reached its highest enrichment by day 6. Measurements of N2 fixation and denitrification indicated an insignificant gain or loss of N from the experimental system by these processes. The data show that woody debris in stream ecosystems might function as a rapid and efficient sink for exogenous N, resulting in stimulation of wood decomposition and subsequent activation of other N cycling processes. PMID:16346785

  8. Extension of transverse relaxation-optimized spectroscopy techniques to allosteric proteins: CO- and paramagnetic fluoromet-hemoglobin [beta (15N-valine)].

    PubMed

    Nocek, J M; Huang, K; Hoffman, B M

    2000-03-14

    We present the first steps in applying transverse relaxation-optimized spectroscopy (TROSY) techniques to the study of allosterism. Each beta-chain of the hemoglobin (Hb) tetramer has 17 valine residues. We have (15)N-labeled the beta-chain Val residues and detected 16 of the 17 (1)H-(15)N correlation peaks for beta-chain Val of the R state CO-Hb structure by using the TROSY technique. Sequence-specific assignments are suggested, based mainly on analysis of the (1)H pseudocontact-shift increments produced by oxidizing the diamagnetic R state HbCO to the paramagnetic R state fluoromet form. When possible, we support these assignments with sequential nuclear Overhauser effect (NOE) information obtained from a two-dimensional [(1)H,(1)H]-NOESY-TROSY experiment (NOESY, NOE spectroscopy). We have induced further the R-T conformational change by adding the allosteric effector, inositol hexaphosphate, to the fluoromet-Hb sample. This change induces substantial increments in the (1)H and (15)N chemical shifts, and we discuss the implication of these findings in the context of the tentative sequence assignments. These preliminary results suggest that amide nitrogen and amide proton chemical shifts in a selectively labeled sample are site-specific probes for monitoring the allosteric response of the ensemble-averaged solution structure of Hb. More important, the chemical-shift dispersion obtained is adequate to permit a complete assignment of the backbone (15)N/(13)C resonances upon nonselective labeling. PMID:10716987

  9. Human Vitamin B12 Absorption and Metabolism are Measured by Accelerator Mass Spectrometry Using Specifically Labeled 14C-Cobalamin

    SciTech Connect

    Carkeet, C; Dueker, S R; Lango, J; Buchholz, B A; Miller, J W; Green, R; Hammock, B D; Roth, J R; Anderson, P J

    2006-01-26

    There is need for an improved test of human ability to assimilate dietary vitamin B{sub 12}. Assaying and understanding absorption and uptake of B{sub 12} is important because defects can lead to hematological and neurological complications. Accelerator mass spectrometry (AMS) is uniquely suited for assessing absorption and kinetics of {sup 14}C-labeled substances after oral ingestion because it is more sensitive than decay counting and can measure levels of carbon-14 ({sup 14}C) in microliter volumes of biological samples, with negligible exposure of subjects to radioactivity. The test we describe employs amounts of B{sub 12} in the range of normal dietary intake. The B{sub 12} used was quantitatively labeled with {sup 14}C at one particular atom of the DMB moiety by exploiting idiosyncrasies of Salmonellametabolism. In order to grow aerobically on ethanolamine, S. entericamust be provided with either pre-formed B{sub 12} or two of its precursors: cobinamide and dimethylbenzimidazole (DMB). When provided with {sup 14}C-DMB specifically labeled in the C2 position, cells produced {sup 14}C-B{sub 12} of high specific activity (2.1 GBq/mmol, 58 mCi/mmol) and no detectable dilution of label from endogenous DMB synthesis. In a human kinetic study, a physiological dose (1.5 mg, 2.2 KBq/59 nCi) of purified {sup 14}C-B{sub 12} was administered and showed plasma appearance and clearance curves consistent with the predicted behavior of the pure vitamin. This method opens new avenues for study of B{sub 12} assimilation.

  10. Metabolism of nonessential N-15-labeled amino acids and the measurement of human whole-body protein synthesis rates

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Settle, R. G.; Albina, J. A.; Melnick, G.; Dempsey, D. T.

    1991-01-01

    Eight N-15-labeled nonessential amino acids plus (N-15)H4Cl were administered over a 10-h period to four healthy adult males using a primed-constant dosage regimen. The amount of N-15 excreted in the urine and the urinary ammonia, hippuric acid, and plasma alanine N-15 enrichments were measured. There was a high degree of consistency across subjects in the ordering of the nine compounds based on the fraction of N-15 excreted.

  11. The effect of noncollinearity of 15N-1H dipolar and 15N CSA tensors and rotational anisotropy on 15N relaxation, CSA/dipolar cross correlation, and TROSY.

    PubMed

    Fushman, D; Cowburn, D

    1999-02-01

    Current approaches to 15N relaxation in proteins assume that the 15N-1H dipolar and 15N CSA tensors are collinear. We show theoretically that, when there is significant anisotropy of molecular rotation, different orientations of the two tensors, experimentally observed in proteins, nucleic acids, and small peptides, will result in differences in site-specific correlation functions and spectral densities. The standard treatments of the rates of longitudinal and transverse relaxation of amide 15N nuclei, of the 15N CSA/15N-1H dipolar cross correlation, and of the TROSY experiment are extended to account for the effect of noncollinearity of the 15N-1H dipolar and 15N CSA (chemical shift anisotropy) tensors. This effect, proportional to the degree of anisotropy of the overall motion, (D parallel/D perpendicular - 1), is sensitive to the relative orientation of the two tensors and to the orientation of the peptide plane with respect to the diffusion coordinate frame. The effect is negligible at small degrees of anisotropy, but is predicted to become significant for D parallel/D perpendicular > or = 1.5, and at high magnetic fields. The effect of noncollinearity of 15N CSA and 15N-1H dipolar interaction is sensitive to both gross (hydrodynamic) properties and atomic-level details of protein structure. Incorporation of this effect into relaxation data analysis is likely to improve both precision and accuracy of the derived characteristics of protein dynamics, especially at high magnetic fields and for molecules with a high degree of anisotropy of the overall motion. The effect will also make TROSY efficiency dependent on local orientation in moderately anisotropic systems. PMID:10070755

  12. Retrospective characterization of ontogenetic shifts in killer whale diets via δ13C and δ15N analysis of teeth

    USGS Publications Warehouse

    Newsome, Seth D.; Etnier, Michael A.; Monson, Daniel H.; Fogel, Marilyn L.

    2009-01-01

    Metabolically inert, accretionary structures such as the dentin growth layers in teeth provide a life history record of individual diet with near-annual resolution. We constructed ontogenetic δ13C and δ15N profiles by analyzing tooth dentin growth layers from 13 individual killer whales Orcinus orca collected in the eastern northeast Pacific Ocean between 1961 and 2003. The individuals sampled were 6 to 52 yr old, representing 2 ecotypes—resident and transient—collected across ~25° of latitude. The average isotopic values of transient individuals (n = 10) are consistent with a reliance on mammalian prey, while the average isotopic values of residents (n = 3) are consistent with piscivory. Regardless of ecotype, most individuals show a decrease in δ15N values of ~2.5‰ through the first 3 yr of life, roughly equivalent to a decrease of one trophic level. We interpret this as evidence of gradual weaning, after which, ontogenetic shifts in isotopic values are highly variable. A few individuals (n = 2) maintained relatively stable δ15N and δ13C values throughout the remainder of their lives, whereas δ15N values of most (n = 11) increased by ~1.5‰, suggestive of an ontogenetic increase in trophic level. Significant differences in mean δ13C and δ15N values among transients collected off California suggest that individuality in prey preferences may be prevalent within this ecotype. Our approach provides retrospective individual life history and dietary information that cannot be obtained through traditional field observations of free-ranging and elusive species such as killer whales, including unique historic ecological information that pre-dates modern studies. By providing insights into individual diet composition, stable isotope analysis of teeth and/or bones may be the only means of evaluating a number of hypothesized historical dietary shifts in killer whales of the northeast Pacific Ocean

  13. Retrospective characterization of ontogenetic shifts in killer whale diets via δ13C and δ15N analysis of teeth

    USGS Publications Warehouse

    Newsome, Seth D.; Etnier, Michael A.; Monson, Daniel H.; Fogel, Marilyn L.

    2009-01-01

    Metabolically inert, accretionary structures such as the dentin growth layers in teeth provide a life history record of individual diet with near-annual resolution. We constructed ontogenetic ??13C and ??15N profiles by analyzing tooth dentin growth layers from 13 individual killer whales Orcinus orca collected in the eastern northeast Pacific Ocean between 1961 and 2003. The individuals sampled were 6 to 52 yr old, representing 2 ecotypes-resident and transient - collected across ???25?? of latitude. The average isotopic values of transient individuals (n = 10) are consistent with a reliance on mammalian prey, while the average isotopic values of residents (n = 3) are consistent with piscivory. Regardless of ecotype, most individuals show a decrease in ??15N values of ???2.5% through the first 3 yr of life, roughly equivalent to a decrease of one trophic level. We interpret this as evidence of gradual weaning, after which, ontogenetic shifts in isotopic values are highly variable. A few individuals (n = 2) maintained relatively stable ??15N and ??13C values throughout the remainder of their lives, whereas ??15N values of most (n = 11) increased by ???1.5%, suggestive of an ontogenetic increase in trophic level. Significant differences in mean ??13C and ??15N values among transients collected off California suggest that individuality in prey preferences may be prevalent within this ecotype. Our approach provides retrospective individual life history and dietary information that cannot be obtained through traditional field observations of free-ranging and elusive species such as killer whales, including unique historic ecological information that pre-dates modern studies. By providing insights into individual diet composition, stable isotope analysis of teeth and/or bones may be the only means of evaluating a number of hypothesized historical dietary shifts in killer whales of the northeast Pacific Ocean. ?? Inter-Research 2009.

  14. EFFECT OF DIETARY CLA ON METABOLISM OF ISOTOPE-LABELED OLEIC, LINOLEIC AND CONJUGATED LINOLEIC ACID ISOMERS IN WOMEN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Animal studies suggest that conjugated linoleic acid (CLA) has potential health benefits, but no definitive metabolic data are available to support the possibility that it has physiological activity in humans. The purpose of this study was to determine the effect of dietary CLA on oleic, linoleic, ...

  15. An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes*

    PubMed Central

    MacLeod, A. Kenneth; Fallon, Padraic G.; Sharp, Sheila; Henderson, Colin J.; Wolf, C. Roland; Huang, Jeffrey T.-J.

    2015-01-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

  16. Lysine and protein metabolism in young women. Subdivision based on the novel use of multiple stable isotopic labels.

    PubMed Central

    Irving, C S; Thomas, M R; Malphus, E W; Marks, L; Wong, W W; Boutton, T W; Klein, P D

    1986-01-01

    A multitracer stable isotope study of lysine kinetics was carried out in fasted adult female volunteers to determine whether a multicompartmental model that partitions protein synthesis and breakdown into at least two types of tissue components can be constructed from plasma and breath data. Five female subjects, maintained on formula diets, received L-[13C1]lysine (27 mumol/kg) as an i.v. bolus and L-[15N2]lysine (27 mumol/kg) as an oral bolus 4 h postprandially. Plasma and breath samples were collected for 6 h. On an alternate day, subjects received NaH13CO3 (10 mumol/kg) as an i.v. bolus and breath samples were collected for 6 h. Plasma tracer lysine levels were determined by gas chromatography-mass spectrometry isotope ratiometry, and breath 13CO2 levels were measured by mass spectrometric gas isotope ratiometry. The tracer data could be fitted to a mammillary multicompartmental model that consisted of a lysine central compartment and slow- and fast-exchanging peripheral compartments containing 37, 38, and 324 mumol/kg, respectively. The rates of lysine oxidation, incorporation into protein, and release by protein breakdown were 21, 35, and 56 mmol/kg/h, respectively, in the fast-exchanging compartment, whereas the rates of protein synthesis and breakdown in the slow compartment were both 53 mmol/kg/min. These values corresponded to a whole-body lysine flux of 106 mmol/kg/h. The kinetic parameters were in excellent agreement with reported values obtained by constant-infusion methods. The measurements indicated that it will be possible to detect changes in amino acid pool sizes and protein synthesis and breakdown associated with the mobilization of protein stores from plasma and breath measurements in multitracer stable isotope experiments. PMID:3082937

  17. Differentiation of histidine tautomeric states using (15)N selectively filtered (13)C solid-state NMR spectroscopy.

    PubMed

    Miao, Yimin; Cross, Timothy A; Fu, Riqiang

    2014-08-01

    The histidine imidazole ring in proteins usually contains a mixture of three possible tautomeric states (two neutral - τ and π states and a charged state) at physiological pHs. Differentiating the tautomeric states is critical for understanding how the histidine residue participates in many structurally and functionally important proteins. In this work, one dimensional (15)N selectively filtered (13)C solid-state NMR spectroscopy is proposed to differentiate histidine tautomeric states and to identify all (13)C resonances of the individual imidazole rings in a mixture of tautomeric states. When (15)N selective 180° pulses are applied to the protonated or non-protonated nitrogen region, the (13)C sites that are bonded to the non-protonated or protonated nitrogen sites can be identified, respectively. A sample of (13)C, (15)N labeled histidine powder lyophilized from a solution at pH 6.3 has been used to illustrate the usefulness of this scheme by uniquely assigning resonances of the neutral τ and charged states from the mixture. PMID:25026459

  18. Differentiation of Histidine Tautomeric States using 15N Selectively Filtered 13C Solid-State NMR Spectroscopy

    PubMed Central

    Miao, Yimin; Cross, Timothy A.; Fu, Riqiang

    2014-01-01

    The histidine imidazole ring in proteins usually contains a mixture of three possible tautomeric states (two neutral - τ and π states and a charged state) at physiological pHs. Differentiating the tautomeric states is critical for understanding how the histidine residue participates in many structurally and functionally important proteins. In this work, one dimensional 15N selectively filtered 13C solid-state NMR spectroscopy is proposed to differentiate histidine tautomeric states and to identify all 13C resonances of the individual imidazole rings in a mixture of tautomeric states. When 15N selective 180° pulses are applied to the protonated or non-protonated nitrogen region, the 13C sites that are bonded to the non-protonated or protonated nitrogen sites can be identified, respectively. A sample of 13C,15N labeled histidine powder lyophilized from a solution at pH 6.3 has been used to illustrate the usefulness of this scheme by uniquely assigning resonances of the neutral τ and charged states from the mixture. PMID:25026459

  19. Differentiation of histidine tautomeric states using 15N selectively filtered 13C solid-state NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Miao, Yimin; Cross, Timothy A.; Fu, Riqiang

    2014-08-01

    The histidine imidazole ring in proteins usually contains a mixture of three possible tautomeric states (two neutral - τ and π states and a charged state) at physiological pHs. Differentiating the tautomeric states is critical for understanding how the histidine residue participates in many structurally and functionally important proteins. In this work, one dimensional 15N selectively filtered 13C solid-state NMR spectroscopy is proposed to differentiate histidine tautomeric states and to identify all 13C resonances of the individual imidazole rings in a mixture of tautomeric states. When 15N selective 180° pulses are applied to the protonated or non-protonated nitrogen region, the 13C sites that are bonded to the non-protonated or protonated nitrogen sites can be identified, respectively. A sample of 13C, 15N labeled histidine powder lyophilized from a solution at pH 6.3 has been used to illustrate the usefulness of this scheme by uniquely assigning resonances of the neutral τ and charged states from the mixture.

  20. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose.

    PubMed

    Thorn, K A; Kennedy, K R

    2002-09-01

    The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined. PMID:12322752

  1. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose

    USGS Publications Warehouse

    Thorn, K.A.; Kennedy, K.R.

    2002-01-01

    The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined.

  2. The measurement of sequential changes in cerebral blood flow and oxygen metabolism by positron computed tomography with continuous inhalation of oxygen-15 labeled gases

    SciTech Connect

    Tanada, S.; Yonekura, Y.; Senda, M.; Nishimura, K.; Tamaki, N.; Saji, H.; Fujita, T.; Kobayashi, A.; Taki, W.; Ishikawa, M.

    1984-01-01

    The use of continuous inhalation of oxygen-15 labeled gases is a widely accepted method to measure regional cerebral blood flow (CBF) and oxygen metabolism (CMRO/sub 2/) with positron computed tomography (PCT). The purpose of this study is to evaluate the feasibility to measure sequential changes in CBF and CMRO/sub 2/ by PCT. The functional images of CBF, oxygen extraction fraction (OEF), and CMRO/sub 2/ were obtained using continuous inhalation of oxygen-15 labeled carbon dioxide and oxygen. The effects of spinal drainage in CBF and CMRO/sub 2/ were studied in patients with hydrocephalus following subarachnoid hemorrhage due to the rupture of intracranial aneurysm. Following the measurement in control state, 20 ml of cerebrospinal fluid (CSF) were withdrawn gradually through lumbar puncture, and sequential PCT scans were performed. CBF and CMRO/sub 2/ were markedly depressed in the case with hydrocephalus. The drainage of CSF significantly improved OEF and CMRO/sub 2/, whereas CBF remained depressed. In patients with chronic cerebrovascular disease, the changes in CBF were studied with inhalation of 5% carbon dioxide (CO/sub 2/). CO/sub 2/ loading demonstrated the increase in CBF, while poor regional increase was observed in ''moyamoya'' disease, which permitted the assessment of vascular response to the elevation of plasma CO/sub 2/. The authors preliminary work indicated the potential usefulness of sequential PCT to study the changes in CBF and CMRO/sub 2/ with various interventions.

  3. Metabolism of 3H- and 14C-labeled glutamate, proline, and alanine in normal and adrenalectomized rats using different sites of tracer administration and sampling.

    PubMed

    Said, H M; Chenoweth, M; Dunn, A

    1989-08-01

    Alanine, glutamate and proline labeled with 14C and 3H were infused into fasted normal and adrenalectomized rats. Alanine was administered by the A-V mode (arterial administration-venous sampling), and glutamate and proline by both the A-V and V-A (venous administration-arterial sampling) modes. The kinetics of 14C alanine and 14C glutamate differed markedly from those of the tritium-labeled compounds, but there was little difference in the kinetics of 3H and 14C proline. The replacement rate calculated from the A-V mode for glutamate was about half that obtained in the V-A mode, but there was little difference with proline. The masses of the amino acids (total content of amino acids in the body) were calculated from the washout curves of the tritium-labeled compounds after the infusion of tracer was terminated. The masses for the normal rats were 407 mumol/kg for alanine, 578 mumol/kg for glutamate and 296 mumol/kg for proline. The so-called distribution spaces calculated conventionally from total masses and the amino acid concentrations in plasma are much greater than the volume of the body, reflecting the fact that amino acid concentrations in tissues greatly exceed those in plasma. Adrenalectomy markedly affected the kinetics of the three amino acids, and their replacement rates were greatly reduced. The proline and glutamate masses were reduced by at least one half, while that of alanine was unchanged. Adrenalectomy markedly reduced the conversion of proline to glutamate. The hydrocortisone regimen used in this study restored the metabolism of alanine and glutamate to normal, but had no effect on that of proline. PMID:2569659

  4. Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis.

    PubMed

    Gaudin, Alice; Lepetre-Mouelhi, Sinda; Mougin, Julie; Parrod, Martine; Pieters, Grégory; Garcia-Argote, Sébastien; Loreau, Olivier; Goncalves, Jordan; Chacun, Hélène; Courbebaisse, Yann; Clayette, Pascal; Desmaële, Didier; Rousseau, Bernard; Andrieux, Karine; Couvreur, Patrick

    2015-08-28

    Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury. PMID:26087468

  5. Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis

    PubMed Central

    Gaudin, Alice; Lepetre-Mouelhi, Sinda; Mougin, Julie; Parrod, Martine; Pieters, Grégory; Garcia-Argote, Sébastien; Loreau, Olivier; Goncalves, Jordan; Chacun, Hélène; Courbebaisse, Yann; Clayette, Pascal; Desmaële, Didier; Rousseau, Bernard; Andrieux, Karine; Couvreur, Patrick

    2015-01-01

    Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[3H]-Adenosine NAs and [14C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1 h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury. PMID:26087468

  6. 15N2 formation and fast oxygen isotope exchange during pulsed 15N18O exposure of MnOx/CeO2

    SciTech Connect

    Kwak, Ja Hun; Szanyi, Janos

    2014-12-23

    Pulsing 15N18O onto an annealed 1% Mn16Ox/Ce16O2 catalyst resulted in very fast oxygen isotope exchange and 15N2 formation at 295 K. In the 1st 15N18O pulse, due to the presence of large number of surface oxygen defects, extensive 15N218O and 15N2 formations were observed. In subsequent pulses oxygen isotope exchange dominated as a result of highly labile oxygen in the oxide. We gratefully acknowledge the US Department of Energy (DOE), Office of Energy Efficiency and Renewable Energy/Vehicle Technologies Program for the support of this work. The research described in this paper was performed at the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is operated for the US DOE by Battelle.

  7. Resolving the bulk δ 15N values of ancient human and animal bone collagen via compound-specific nitrogen isotope analysis of constituent amino acids

    NASA Astrophysics Data System (ADS)

    Styring, Amy K.; Sealy, Judith C.; Evershed, Richard P.

    2010-01-01

    the biosynthetic pathway of threonine and the other amino acids. The δ 15N value of phenylalanine does not change significantly with trophic level, reflecting its conservative nature as an essential amino acid, and thus represents the isotopic composition of the nitrogen at the base of the food web. Δ 15N Glu-Phe values in particular are shown to reflect trophic level nitrogen sources within a food web. In relation to the reconstruction of ancient human diet the contribution of marine and terrestrial protein are strongly reflected in Δ 15N Glu-Phe values. Differences in nitrogen metabolism are also shown to have an influence upon individual amino acid δ 15N values with Δ 15N Glu-Phe values emphasising differences between the different physiological adaptations. The latter is demonstrated in tortoises, which can excrete nitrogen in the form of uric acid and urea and display negative Δ 15N Glu-Phe values whereas those for marine and terrestrial mammals are positive. The findings amplify the potential advantages of compound-specific nitrogen isotope analysis in the study of nitrogen flow within food webs and in the reconstruction of past human diets.

  8. Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study.

    PubMed

    Sahu, S C; Bhuyan, A K; Udgaonkar, J B; Hosur, R V

    2000-10-01

    Backbone dynamics of uniformly 15N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 degrees C, pH 6.6, using 15N relaxation data obtained from proton-detected 2D [1H]-15N NMR spectroscopy. 15N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and steady-state heteronuclear [1H]-15N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide 15N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (tau(m)) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motion's cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme action. PMID

  9. Label-free surface-enhanced Raman scattering imaging to monitor the metabolism of antitumor drug 6-mercaptopurine in living cells.

    PubMed

    Han, Guangmei; Liu, Renyong; Han, Ming-Yong; Jiang, Changlong; Wang, Jianping; Du, Shuhu; Liu, Bianhua; Zhang, Zhongping

    2014-12-01

    The molecular processes of drugs from cellular uptake to intracellular distribution as well as the intracellular interaction with the target molecule are critically important for the development of new antitumor drugs. In this work, we have successfully developed a label-free surface-enhanced Raman scattering (SERS) technique to monitor and visualize the metabolism of antitumor drug 6-mercaptopurine in living cells. It has been clearly demonstrated that Au@Ag NPs exhibit an excellent Raman enhancement effect to both 6-mercaptopurine and its metabolic product 6-mercaptopurine-ribose. Their different ways to absorb at the surface of Au@Ag NPs lead to the obvious spectral difference for distinguishing the antitumor drug and its metabolite by SERS spectra. The Au@Ag NPs can easily pass through cell membranes in a large amount and sensitively respond to the biological conversion of 6-mercaptopurine in tumor cells. The Raman imaging can visualize the real-time distribution of 6-mercaptopurine and its biotransformation with the concentrations in tumor cells. The SERS-based method reported here is simple and efficient for the assessments of drug efficacy and the understanding of the molecular therapeutic mechanism of antitumor drugs at the cellular level. PMID:25372629

  10. Potential use of carbon-11 labeled alpha-aminoisobutyric acid (AIB) as an in vivo tracer of amino acid uptake in differing metabolic states

    SciTech Connect

    Conti, P.S.; Starnes, H.F.; Brennan, M.F.

    1986-05-01

    AIB has been used as a model amino acid for the evaluation of alanine-preferring amino acid transport. Hormonal factors and starvation alter the tissue distribution of amino acids, particularly in liver and muscle. With positron emission tomography and labeling of biochemical tracers with C-11, (t1/2=20.4 min), it is now possible to study amino acid kinetics in vivo using external imaging. In order to investigate the utility of C-11 AIB as an in vivo tracer of altered tissue metabolism, C-14 AIB was studied in groups of rats with either streptozotocin-induced diabetes, insulin-induced hypoglycemia or starvation. The data suggest an increased amino acid uptake in liver in starvation, an increased uptake in muscle in response to insulin and associated hypoglycemia and decreased transport in muscle in starvation, as seen by other investigators. These results suggest that C-11 AIB may be useful as an in vivo monitor of metabolic changes in body tissues.

  11. Fate of nitrogen deposition and decomposed nitrogen from litter in a 15N-tracer mesocosm experiment

    NASA Astrophysics Data System (ADS)

    Nair, R.; Perks, M.; Mencuccini, M.

    2013-12-01

    Atmospheric deposition of anthropogenic-derived nitrogen may be a major driver of the 0.6-0.7 Pg y-1 increase in the carbon sink in historically N-limited northern and boreal forests, but the magnitude of its effect is still uncertain. A strong effect depends on the allocation of N to trees, because of their high C:N ratio in woody tissues, and isotope tracer experiments have shown that the majority of 15N tracers applied directly to the soil are lost via leeching or retained in soil pools rather than being acquired by tree root systems. However, ambient anthropogenic inputs of N to these systems are transported in the atmosphere and intercepted by foliage before they reach the soil system, while labelled fertilization experiments also can only explicitly trace the fate of the 15N-tracer from deposition, as opposed to changes in the fate of N from litter, where decomposition rates may be enhanced at low ambient levels of deposition, affecting the availability of N from this pool for tree nutrition. We present initial results from a potted Sitka Spruce mesocosm 15N-tracer experiment where ambient nitrogen deposition was supplemented with a minor (0.4 kg ha-1 y-1) input of additional N, applied to either the soil or the foliage. Either this deposition, or litter in the pots, was enriched in 15N, allowing the fate of the isotope from two different methods of deposition to be compared with that of nitrogen released from the litter under the deposition treatment.

  12. Delivery-Corrected Imaging of Fluorescently-Labeled Glucose Reveals Distinct Metabolic Phenotypes in Murine Breast Cancer

    PubMed Central

    Frees, Amy E.; Rajaram, Narasimhan; McCachren, Samuel S.; Fontanella, Andrew N.; Dewhirst, Mark W.; Ramanujam, Nimmi

    2014-01-01

    When monitoring response to cancer therapy, it is important to differentiate changes in glucose tracer uptake caused by altered delivery versus a true metabolic shift. Here, we propose an optical imaging method to quantify glucose uptake and correct for in vivo delivery effects. Glucose uptake was measured using a fluorescent D-glucose derivative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-D-glucose (2-NBDG) in mice implanted with dorsal skin flap window chambers. Additionally, vascular oxygenation (SO2) was calculated using only endogenous hemoglobin contrast. Results showed that the delivery factor proposed for correction, “RD”, reported on red blood cell velocity and injected 2-NBDG dose. Delivery-corrected 2-NBDG uptake (2-NBDG60/RD) inversely correlated with blood glucose in normal tissue, indicating sensitivity to glucose demand. We further applied our method in metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. The ratio 2-NBDG60/RD was increased in 4T1 tumors relative to 4T07 tumors yet average SO2 was comparable, suggesting a shift toward a “Warburgian” (aerobic glycolysis) metabolism in the metastatic 4T1 line. In heterogeneous regions of both 4T1 and 4T07, 2-NBDG60/RD increased slightly but significantly as vascular oxygenation decreased, indicative of the Pasteur effect in both tumors. These data demonstrate the utility of delivery-corrected 2-NBDG and vascular oxygenation imaging for differentiating metabolic phenotypes in vivo. PMID:25526261

  13. Metabolism of Nonessential N15-Labeled Amino Acids and the Measurement of Human Whole-Body Protein Synthesis Rates

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Settle, R. G.; Albina, J. A.; Dempsey, D. T.; Melnick, G.

    1991-01-01

    Eight N-15 labeled nonessential amino acids plus (15)NH4Cl were administered over a 10 h period to four healthy adult males using a primed-constant dosage regimen. The amount of N-15 excreted in the urine and the urinary ammonia, hippuric acid, and plasma alanine N-15 enrichments were measured. There was a high degree of consistency across subjects in the ordering of the nine compounds based on the fraction of N-15 excreted (Kendall coefficient of concordance W = 0.83, P is less than 0.01). Protein synthesis rates were calculated from the urinary ammonia plateau enrichment and the cumulative excretion of N-15. Glycine was one of the few amino acids that gave similar values by both methods.

  14. High levels of isotope elimination improve precision and allow individual-based measurements of metabolic rates in animals using the doubly labeled water method

    PubMed Central

    Shirai, Masaki; Niizuma, Yasuaki; Yamamoto, Maki; Oda, Emiko; Ebine, Naoyuki; Oka, Nariko; Yoda, Ken

    2015-01-01

    Doubly labeled water (DLW) can be used to measure energy expenditure in free-ranging animals, but questions have been raised about its accuracy in different species or contexts. We investigated whether differences in the extent of isotope elimination affects the precision and accuracy of the DLW method, which can vary according to the experimental design or metabolic rate of the species. Estimated total energy expenditure by the DLW method (TEEdlw) was compared with actual total energy expenditure simultaneously measured via respirometry (TEEresp) in streaked shearwaters Calonectris leucomelas, a pelagic seabird. Subjects were divided into three groups with different experimental conditions: at rest on the ground for 24 h (Group A) or for 48 h (Group B), and at rest on the water for 24 h (Group C). TEEdlw in Group A matched TEEresp, whereas there was an overestimation of TEEdlw in both Groups B and C compared with TEEresp. However, compared with Group A, TEEdlw in Groups B and C had reduced the isotopic analytical variability and thus higher precision. The best regression model (TEEdlw = 1.37 TEEresp − 14.12) showed a high correlation (R2 = 0.82) between TEEdlw and TEEresp and allows a correction factor for field metabolic rates in streaked shearwaters. Our results demonstrate that the commonly made assumption that the DLW method is not appropriate for individual-based estimates may be incorrect in certain circumstances. Although a correction factor may be necessary when using the DLW method to estimate metabolic rate, greater levels of isotope eliminations provides DLW estimates with high precision, which can adequately represent relative individual estimates. Nevertheless, the DLW method, should be used with caution when characterizing interspecies difference of energy expenditures. PMID:26611463

  15. Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza.

    PubMed

    Yin, Yuling; Watanabe, Shin; Ashihara, Hiroshi

    2012-01-01

    We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots. PMID:22888538

  16. Dual, differential isotope labeling shows the preferential movement of labile plant constituents into mineral-bonded soil organic matter.

    PubMed

    Haddix, Michelle L; Paul, Eldor A; Cotrufo, M Francesca

    2016-06-01

    The formation and stabilization of soil organic matter (SOM) are major concerns in the context of global change for carbon sequestration and soil health. It is presently believed that lignin is not selectively preserved in soil and that chemically labile compounds bonding to minerals comprise a large fraction of the SOM. Labile plant inputs have been suggested to be the main precursor of the mineral-bonded SOM. Litter decomposition and SOM formation are expected to have temperature sensitivity varying with the lability of plant inputs. We tested this framework using dual (13) C and (15) N differentially labeled plant material to distinguish the metabolic and structural components within a single plant material. Big Bluestem (Andropogon gerardii) seedlings were grown in an enriched (13) C and (15) N environment and then prior to harvest, removed from the enriched environment and allowed to incorporate natural abundance (13) C-CO2 and (15) N fertilizer into the metabolic plant components. This enabled us to achieve a greater than one atom % difference in (13) C between the metabolic and structural components within the plant litter. This differentially labeled litter was incubated in soil at 15 and 35 °C, for 386 days with CO2 measured throughout the incubation. After 14, 28, 147, and 386 days of incubation, the soil was subsequently fractionated. There was no difference in temperature sensitivity of the metabolic and structural components with regard to how much was respired or in the amount of litter biomass stabilized. Only the metabolic litter component was found in the sand, silt, or clay fraction while the structural component was exclusively found in the light fraction. These results support the stabilization framework that labile plant components are the main precursor of mineral-associated organic matter. PMID:27142168

  17. /sup 15/N-Ammonia assimilation, 2-oxoglutarate transport, and glutamate export in spinach chloroplasts in the presence of dicarboxylates in the light

    SciTech Connect

    Woo, K.C.; Boyle, F.A.; Flugge, I.U.; Heldt, H.W.

    1987-11-01

    The direct incorporation of /sup 15/NH/sub 4/Cl into amino acids in illuminated spinach (Spinacia oleracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during /sup 15/NH/sub 4/ assimilation in the presence of malate. In 4 minutes the /sup 15/N-label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the N-atom of aspartate was only 3%. The addition of malate during the assimilation of /sup 15/NH/sub 4/Cl and Na/sup 15/NO/sub 2/ greatly increased the /sup 15/N-label into glutamine but did not qualitatively change the order of the incorporation of /sup 15/N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in (/sup 14/C)2-oxoglutarate transport into the chloroplast as well as an increase in the export of (/sup 14/C)glutamate out of the chloroplast. Little (/sup 14/C)glutamine was detected in the medium of the chloroplast preparations. The stimulation of /sup 15/N-incorporation and (/sup 14/C)glutamate export by malate could be directly attributed to the increase in 2-oxoglutarate transport activity (via the 2-oxoglutarate translocator) observed in the presence of exogenous malate.

  18. An innovative, quick and convenient labeling method for the investigation of pharmacological behavior and the metabolism of poly(DL-lactide-co-glycolide) nanospheres

    NASA Astrophysics Data System (ADS)

    Stevanović, Magdalena; Maksin, Tatjana; Petković, Jana; Filipič, Metka; Uskoković, Dragan

    2009-08-01

    Nanoparticles of poly(DL-lactide-co-glycolide) (PLGA) in the size range 90-150 nm were produced using the physicochemical method with solvent/non-solvent systems. The encapsulation of the ascorbic acid in the polymer matrix was performed by homogenization of the water and organic phases. In vitro degradation and release tests of PLGA nanoparticles with and without encapsulated ascorbic acid were studied for more than 60 days in PBS and it has been determined that PLGA completely degrades within this period, fully releasing all encapsulated ascorbic acid. The cytotoxicity of PLGA and PLGA/ascorbic acid 85/15% nanoparticles was examined with human hepatoma cell lines (HepG2 ECACC), in vitro. The obtained results indicate that neither PLGA nanospheres nor PLGA/ascorbic acid 85/15% nanoparticles significantly affected the viability of the HepG2 cells. The investigation of the distribution and pharmacokinetics of PLGA is crucial for the effective prediction of host responses to PLGA in particular applications. Thus we present a method of labeling PLGA nanospheres and PLGA/ascorbic acid 85/15 wt% nanoparticles by 99mTc which binds outside, leaving the cage intact. This enables a quick and convenient investigation of the pharmacological behavior and metabolism of PLGA. The biodistribution of 99mTc-labeled PLGA particles with and without encapsulated ascorbic acid after different periods of time of their installation into rats was examined. PLGA nanospheres with encapsulated ascorbic acid exhibit prolonged blood circulation accompanied by time-dependent reduction in the lungs, liver and spleen, and addition in the kidney, stomach and intestine. The samples were characterized by x-ray diffraction, scanning electron microscopy, stereological analysis, transmission electron microscopy, ultraviolet spectroscopy and instant thin layer chromatography.

  19. Validity of using a 3-dimensional PET scanner during inhalation of 15O-labeled oxygen for quantitative assessment of regional metabolic rate of oxygen in man

    NASA Astrophysics Data System (ADS)

    Hori, Yuki; Hirano, Yoshiyuki; Koshino, Kazuhiro; Moriguchi, Tetsuaki; Iguchi, Satoshi; Yamamoto, Akihide; Enmi, Junichiro; Kawashima, Hidekazu; Zeniya, Tsutomu; Morita, Naomi; Nakagawara, Jyoji; Casey, Michael E.; Iida, Hidehiro

    2014-09-01

    Use of 15O labeled oxygen (15O2) and positron emission tomography (PET) allows quantitative assessment of the regional metabolic rate of oxygen (CMRO2) in vivo, which is essential to understanding the pathological status of patients with cerebral vascular and neurological disorders. The method has, however, been challenging, when a 3D PET scanner is employed, largely attributed to the presence of gaseous radioactivity in the trachea and the inhalation system, which results in a large amount of scatter and random events in the PET assessment. The present study was intended to evaluate the adequacy of using a recently available commercial 3D PET scanner in the assessment of regional cerebral radioactivity distribution during an inhalation of 15O2. Systematic experiments were carried out on a brain phantom. Experiments were also performed on a healthy volunteer following a recently developed protocol for simultaneous assessment of CMRO2 and cerebral blood flow, which involves sequential administration of 15O2 and C15O2. A particular intention was to evaluate the adequacy of the scatter-correction procedures. The phantom experiment demonstrated that errors were within 3% at the practically maximum radioactivity in the face mask, with the greatest radioactivity in the lung. The volunteer experiment demonstrated that the counting rate was at peak during the 15O gas inhalation period, within a verified range. Tomographic images represented good quality over the entire FOV, including the lower part of the cerebral structures and the carotid artery regions. The scatter-correction procedures appeared to be important, particularly in the process to compensate for the scatter originating outside the FOV. Reconstructed images dramatically changed if the correction was carried out using inappropriate procedures. This study demonstrated that accurate reconstruction could be obtained when the scatter compensation was appropriately carried out. This study also suggested the

  20. Variation in Growth, Colonization of Maize, and Metabolic Parameters of GFP- and DsRed-Labeled Fusarium verticillioides Strains.

    PubMed

    Wu, Lei; Conner, R L; Wang, Xiaoming; Xu, Rongqi; Li, Hongjie

    2016-08-01

    Autofluorescent proteins are frequently applied as visual markers in the labeling of filamentous fungi. Genes gfp and DsRed were transformed into the genome of Fusarium verticillioides via the Agrobacterium tumefaciens-mediated transformation method. The selected transformants displayed a bright green or red fluorescence in all the organelles of the growing fungal mycelia and spores (except for the vacuoles) both in cultures and in the maize (Zea mays) roots they colonized. The results of gene-specific polymerase chain reaction (PCR) analysis and the thermal asymmetrical interlaced (TAIL)-PCR analysis demonstrated that gfp and DsRed were integrated on different chromosomes of the fungus. Reductions in the colony growth on the plates at pH 4.0 and 5.5 was observed for the green fluorescent protein (GFP)-transformant G3 and the DsRed-transformant R4, but transformants G4 and R1 grew as well as the wild-type strain at pH 4.0. The speed of growth of all the transformants was similar to the wild-type strain at pH ≥ 7. The insertion of gfp and DsRed did not alter the production of extracellular enzymes and fumonisin B by F. verticillioides. The transformants expressing GFP and DsRed proteins were able to colonize maize roots. However, the four transformants examined produced fewer CFU in the root samples than the wild-type strain during a sampling period of 7 to 28 days after inoculation. PMID:27088391

  1. 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. PMID:26649387

  2. Mid-late Holocene North Pacific trade winds as inferred from deep-sea coral skeletal δ15N and δ13C biogeochemistry

    NASA Astrophysics Data System (ADS)

    Guilderson, T. P.; Mccarthy, M. D.; Dunbar, R. B.; Englebrecht, A.; Roark, E.

    2013-12-01

    δ15N and δ13C data obtained from samples of proteinaceous deep-sea corals collected from the North Pacific Subtropical Gyre (Hawai'ian archipelago) and the central equatorial Pacific (Line Islands) document multi-decadal to century scale variability in the isotopic composition of surface-produced particulate organic matter exported to the deep sea. Comparison of the δ13C data, where Line Island samples are 0.6‰ more positive than the Hawai'ian samples, support the contention that the North Pacific Subtropical Gyre is more efficient than the tropical upwelling system at trapping and/or recycling nutrients within the mixed layer. δ15N values from the Line Island samples are also more positive than those from the central gyre, and within the Hawai'ian samples there is a gradient with more positive δ15N values in samples from the main Hawai'ian Islands versus French Frigate Shoals in the Northwestern Hawai'ian Islands. The gradient in the Hawai'ian samples likely reflects the relative importance of algal acquisition of metabolic N via dissolved seawater nitrate uptake versus nitrogen fixation. The Hawai'ian sample set also exhibits a strong decrease in δ15N values from the mid-Holocene to present. We hypothesize that this decrease is most likely the result of decreasing tradewinds, and possibly a commensurate decrease in entrainment of more positive δ15N-NO3 subthermocline water masses.

  3. Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies

    SciTech Connect

    Modi, S.; Behere, D.V.; Mitra, S. )

    1989-05-30

    The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 +/- 5, 173 +/- 20, and 83 +/- 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 +/- 0.2 A from the 15N T1 measurements.

  4. Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

    PubMed Central

    Ruzsics, Zsolt; Friedel, Caroline C.; Koszinowski, Ulrich H.; Dölken, Lars

    2013-01-01

    The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated. We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms. PMID:23963265

  5. Factors Controlling the Stable Nitrogen Isotopic Composition (δ15N) of Lipids in Marine Animals.

    PubMed

    Svensson, Elisabeth; Schouten, Stefan; Hopmans, Ellen C; Middelburg, Jack J; Sinninghe Damsté, Jaap S

    2016-01-01

    Lipid extraction of biomass prior to stable isotope analysis is known to cause variable changes in the stable nitrogen isotopic composition (δ15N) of residual biomass. However, the underlying factors causing these changes are not yet clear. Here we address this issue by comparing the δ15N of bulk and residual biomass of several marine animal tissues (fish, crab, cockle, oyster, and polychaete), as well as the δ15N of the extracted lipids. As observed previously, lipid extraction led to a variable offset in δ15N of biomass (differences ranging from -2.3 to +1.8 ‰). Importantly, the total lipid extract (TLE) was highly depleted in 15N compared to bulk biomass, and also highly variable (differences ranging from -14 to +0.7 ‰). The TLE consisted mainly of phosphatidylcholines, a group of lipids with one nitrogen atom in the headgroup. To elucidate the cause for the 15N-depletion in the TLE, the δ15N of amino acids was determined, including serine because it is one of the main sources of nitrogen to N-containing lipids. Serine δ15N values differed by -7 to +2 ‰ from bulk biomass δ15N, and correlated well with the 15N depletion in TLEs. On average, serine was less depleted (-3‰) than the TLE (-7 ‰), possibly due to fractionation during biosynthesis of N-containing headgroups, or that other nitrogen-containing compounds, such as urea and choline, or recycled nitrogen contribute to the nitrogen isotopic composition of the TLE. The depletion in 15N of the TLE relative to biomass increased with the trophic level of the organisms. PMID:26731720

  6. Factors Controlling the Stable Nitrogen Isotopic Composition (δ15N) of Lipids in Marine Animals

    PubMed Central

    Svensson, Elisabeth; Schouten, Stefan; Hopmans, Ellen C.; Middelburg, Jack J.; Sinninghe Damsté, Jaap S.

    2016-01-01

    Lipid extraction of biomass prior to stable isotope analysis is known to cause variable changes in the stable nitrogen isotopic composition (δ15N) of residual biomass. However, the underlying factors causing these changes are not yet clear. Here we address this issue by comparing the δ15N of bulk and residual biomass of several marine animal tissues (fish, crab, cockle, oyster, and polychaete), as well as the δ15N of the extracted lipids. As observed previously, lipid extraction led to a variable offset in δ15N of biomass (differences ranging from -2.3 to +1.8 ‰). Importantly, the total lipid extract (TLE) was highly depleted in 15N compared to bulk biomass, and also highly variable (differences ranging from -14 to +0.7 ‰). The TLE consisted mainly of phosphatidylcholines, a group of lipids with one nitrogen atom in the headgroup. To elucidate the cause for the 15N-depletion in the TLE, the δ15N of amino acids was determined, including serine because it is one of the main sources of nitrogen to N-containing lipids. Serine δ15N values differed by -7 to +2 ‰ from bulk biomass δ15N, and correlated well with the 15N depletion in TLEs. On average, serine was less depleted (-3‰) than the TLE (-7 ‰), possibly due to fractionation during biosynthesis of N-containing headgroups, or that other nitrogen-containing compounds, such as urea and choline, or recycled nitrogen contribute to the nitrogen isotopic composition of the TLE. The depletion in 15N of the TLE relative to biomass increased with the trophic level of the organisms. PMID:26731720

  7. Evidence of entropy-driven bistability through (15)N NMR analysis of a temperature- and solvent-induced, chiroptical switching polycarbodiimide.

    PubMed

    Reuther, James F; Novak, Bruce M

    2013-12-26

    The thermo- and solvo-driven chiroptical switching process observed in specific polycarbodiimides occurs in a concerted fashion with large deviations in specific optical rotation (OR) and CD Cotton effect as a consequence of varying populations of two distinct polymer conformations. These two conformations are clearly visible in the (15)N NMR and IR spectra of the (15)N-labeled poly((15)N-(1-naphthyl)-N'-octadecylcarbodiimide) (Poly-3) and poly((15)N-(1-naphthyl)-(15)N'-octadecylcarbodiimide) (Poly-5). Using van't Hoff analysis, the enthalpies and entropies of switching (ΔHswitching; ΔSswitching) were calculated for both Poly-3 and Poly-5 using the relative integrations of both peaks in the (15)N NMR spectra at different temperatures to measure the populations of each state. The chiroptical switching (i.e., transitioning from state A to state B) was found to be an endothermic process (positive ΔHswitching) for both Poly-3 and Poly-5 in all solvents studied, meaning the conformation correlating with the downfield chemical shift (ca. 148 ppm, state B) is the higher enthalpy state. The compensating factor behind this phenomenon has been determined to be the large increase in entropy in CHCl3 as a result of the switching. Herein, we propose that the increased entropy in the system is a direct consequence of increased disorder in the solvent as the switching occurs. Specifically, the chloroform solvent molecules are very ordered around the polymer chains due to favorable solvent-polymer interactions, but as the switching occurs, these interactions become less favorable and disorder results. The same level of solvent disorder is not achieved in toluene, causing the chiroptical switching process to occur at higher temperatures. PMID:24313274

  8. 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

  9. Sources and transformations of N in reclaimed coastal tidelands: evidence from soil δ15N data

    NASA Astrophysics Data System (ADS)

    Kwak, Jin-Hyeob; Choi, Woo-Jung; Lim, Sang-Sun; Lee, Seung-Heon; Lee, Sang-Mo; Chang, Scott X.; Jung, Jae-Woon; Yoon, Kwang-Sik; Choi, Soo-Myung

    2008-01-01

    Electrical conductivity of saturated soil extracts (ECe) in three reclaimed tideland (RTL) soils on the west coast of Korea decreased with time since reclamation, indicating natural desalinization through leaching of salts by precipitation water. Soil N concentration increased with decreasing ECe. With the increase in soil N concentration, the δ15N decreased, likely caused by the input of 15N-depleted N sources. As N2-fixing plant species were found in the oldest RTL, atmospheric N2 fixation likely contributed to the increase in soil N concentration in the oldest RTL. Negative δ15N (-7.1 to -2.0‰) of total inorganic N (NH4 ++NO3 -) and published data on N deposition near the study area indicate that atmospheric N deposition might be another source of N in the RTLs. Meanwhile, the consistently negative δ15N of soil NO3 - excluded N input from chemical fertilizer through groundwater flow as a potential N source, since NO3 - in groundwater generally have a positive δ15N. The patterns of δ15N of NH4 + (+2.3 to +5.1‰) and NO3 - (-9.2 to -5.0‰) suggested that nitrification was an active process that caused 15N enrichment in NH4 + but denitrification was probably minimal which would otherwise have caused 15N enrichment in NO3 -. A quantitative approach on N budget would provide a better understanding of soil N dynamics in the studied RTLs.

  10. Increased Plant Uptake of Nitrogen from 15N Depleted Fertilizer Using Plant Growth-Promoting Rhizobacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. 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…

  12. 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…

  13. Disturbance and topography shape nitrogen availability and δ15N over long-term forest succession

    EPA Science Inventory

    Forest disturbance and long-term succession can promote open N cycling that increases N loss and soil δ15N values. We examined soil and foliar patterns in N and δ15N, and soil N mineralization, across a topographically complex montane forest landscape influenced by human logging ...

  14. δ 15 N constraints on long-term nitrogen balances in temperate forests

    EPA Science Inventory

    Natural abundance δ15N of ecosystems integrates nitrogen (N) inputs and losses, and thus reflects factors that control the long-term development of ecosystem N balances. We here report N and carbon (C) content of forest vegetation and soils, and associated δ15N, across nine Doug...

  15. Over 20% (15)N Hyperpolarization in Under One Minute for Metronidazole, an Antibiotic and Hypoxia Probe.

    PubMed

    Barskiy, Danila A; Shchepin, Roman V; Coffey, Aaron M; Theis, Thomas; Warren, Warren S; Goodson, Boyd M; Chekmenev, Eduard Y

    2016-07-01

    Direct NMR hyperpolarization of naturally abundant (15)N sites in metronidazole is demonstrated using SABRE-SHEATH (Signal Amplification by Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei). In only a few tens of seconds, nuclear spin polarization P(15)N of up to ∼24% is achieved using parahydrogen with 80% para fraction corresponding to P(15)N ≈ 32% if ∼100% parahydrogen were employed (which would translate to a signal enhancement of ∼0.1-million-fold at 9.4 T). In addition to this demonstration on the directly binding (15)N site (using J(2)H-(15)N), we also hyperpolarized more distant (15)N sites in metronidazole using longer-range spin-spin couplings (J(4)H-(15)N and J(5)H-(15)N). Taken together, these results significantly expand the range of molecular structures and sites amenable to hyperpolarization via low-cost parahydrogen-based methods. In particular, hyperpolarized nitroimidazole and its derivatives have powerful potential applications such as direct in vivo imaging of mechanisms of action or hypoxia sensing. PMID:27321159

  16. Synthesis of 5-aryl-4-(2-acetylaminobenzoyl)-1,2,3-triazoles with the /sup 15/N isotope at the terminal positions of the triazole rings and the tautomeric composition

    SciTech Connect

    Kurkovskaya, L.N.; Velezheva, V.S.; Sorokina, I.K.; Dmitrevskaya, L.I.; Zhil'nikov, V.G.

    1988-12-20

    A mixture of 4-(2-acetylaminobenzoyl)-5-phenyl(p-cumenyl)-1-/sup 15/N,2,3- and 4-(2-acetylaminobenzoyl)-5-phenyl(p-cumenyl)-1,2,3-/sup 15/N-triazoles was obtained from 1-acetyl-2-arylmethylene-3-indolinones and Na/sup 15/N/sub 3/ with the label at the terminal position. The tautomeric composition of the mixture, which corresponds to a state of equilibrium between the 2H and 3H forms of the triazole ring, was established by /sup 1/H (at low temperatures) and /sup 15/N NMR spectroscopy. The 4-(2-acetylaminobenzoyl)-5-aryl-1,2,3-triazoles are acylated at the sterically less hindered position 2 of the triazole ring.

  17. Symbiotic nitrogen fixation in an arid ecosystem measured by sup 15 N natural abundance

    SciTech Connect

    Johnson, G.V. )

    1990-05-01

    Plants dependent on nitrogen fixation have an {sup 15}N abundance similar to the atmosphere, while non-nitrogen fixing plants usually are enriched in {sup 15}N and are similar to soil nitrogen values. The natural abundance of {sup 15}N in leaf tissues and soils was determined to evaluate symbiotic nitrogen fixation by several legumes and actinorhizal species in the Sevilleta Long-term Ecological Research area in central New Mexico. Comparison of {delta}{sup 15}N values for the legume Prosopis glandulosa (mesquite) to adjacent Atriplex canascens (fourwing saltbush) indicated that P. glandulosa obtained 66% of its nitrogen by fixation. The legume Hoffmanseggia jamesii was found to be utilizing soil nitrogen. The {delta}{sup 15}N values for the actinorhizal plants, Elaeagnus angustifolia and Cercocarpus montanus, while below values for soil nitrogen, did not differ from associated non-fixing plants.

  18. A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases

    PubMed Central

    Jin, Feng; Bhowmik, Salil Kumar; Putluri, Vasanta; Gu, Franklin; Gohlke, Jie; Von Rundstedt, Friedrich Carl; Dasgupta, Subhamoy; Krishnapuram, Rashmi; O’Malley, Bert W.; Sreekumar, Arun; Putluri, Nagireddy

    2016-01-01

    The growth of cancer cells relies more on increased proliferation and autonomy compared to non-malignant cells. The rate of de novo nucleotide biosynthesis correlates with cell proliferation rates. In part, glutamine is needed to sustain high rates of cellular proliferation as a key nitrogen donor in purine and pyrimidine nucleotide biosynthesis. In addition, glutamine serves as an essential substrate for key enzymes involved in the de novo synthesis of purine and pyrimidine nucleotides. Here, we developed a novel liquid chromatography (LC-MS) to quantify glutamine-derived [15N] nitrogen flux into nucleosides and nucleobases (purines and pyrimidines). For this, DNA from 5637 bladder cancer cell line cultured in 15N labelled glutamine and then enzymatically hydrolyzed by sequential digestion. Subsequently, DNA hydrolysates were separated by LC-MS and Selected Reaction Monitoring (SRM) was employed to identify the nucleobases and nucleosides. Thus, high sensitivity and reproducibility of the method make it a valuable tool to identify the nitrogen flux primarily derived from glutamine and can be further adaptable for high throughput analysis of large set of DNA in a clinical setting. PMID:27158554

  19. 15N-Cholamine – A Smart Isotope Tag for Combining NMR- and MS-Based Metabolite Profiling

    PubMed Central

    Tayyari, Fariba; Nagana Gowda, G. A.; Gu, Haiwei; Raftery, Daniel

    2013-01-01

    Recently, the enhanced resolution and sensitivity offered by chemoselective isotope tags have enabled new and enhanced methods for detecting hundreds of quantifiable metabolites in biofluids using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry. However, the inability to effectively detect the same metabolites using both complementary analytical techniques has hindered the correlation of data derived from the two powerful platforms and thereby the maximization of their combined strengths for applications such as biomarker discovery of the identification of unknown metabolites. With the goal of alleviating this bottleneck, we describe a smart isotope tag, 15N-cholamine, which possesses two important properties: an NMR sensitive isotope, and a permanent charge for MS sensitivity. Using this tag, we demonstrate the detection of carboxyl group containing metabolites in both human serum and urine. By combining the individual strengths of the 15N label and permanent charge, the smart isotope tag facilitates effective detection of the carboxyl-containing metabolome by both analytical methods. This study demonstrates a unique approach to exploit the combined strength of MS and NMR in the field of metabolomics. PMID:23930664

  20. Disturbance and topography shape nitrogen availability and δ15 N over long-term forest succession

    USGS Publications Warehouse

    Perakis, Steven; Tepley, Alan J.; Compton, Jana

    2015-01-01

    Forest disturbance and long-term succession towards old-growth are thought to increase nitrogen (N) availability and N loss, which should increase soil δ15N values. We examined soil and foliar patterns in N and δ15N, and soil N mineralization, across 800 years of forest succession in a topographically complex montane landscape influenced by human logging and wildfire. In contrast to expectations, we found that disturbance caused declines in surface mineral soil δ15N values, both in logged forests measured 40–50 years after disturbance, and in unlogged forests disturbed by severe wildfire within the last 200 years. Both symbiotic N fixation and N transfers from disturbed vegetation and detritus could lower soil δ15N values after disturbance. A more important role for symbiotic N fixation is suggested by lower soil δ15N values in slow-successional sites with slow canopy closure, which favors early-successional N fixers. Soil δ15N values increased only marginally throughout 800 years of succession, reflecting soil N uptake by vegetation and strong overall N retention. Although post-disturbance N inputs lowered surface soil δ15N values, steady-state mass balance calculations suggest that wildfire combustion of vegetation and detritus can dominate long-term N loss and increase whole-ecosystem δ15N. On steeper topography, declining soil δ15N values highlight erosion and accelerated soil turnover as an additional abiotic control on N balances. We conclude for N-limited montane forests that soil δ15N and N availability are less influenced by nitrate leaching and denitrification loss than by interactions between disturbance, N fixation, and erosion.

  1. 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. PMID:27010164

  2. Analysis of proteome dynamics in mice by isotopic labeling.

    PubMed

    Price, John C; Ghaemmaghami, Sina

    2014-01-01

    Recent advances in mass spectrometry and in vivo isotopic labeling have enabled proteome-wide analyses of protein turnover in complex organisms. Here, we describe a protocol for analyzing protein turnover rates in mouse tissues by comprehensive (15)N labeling. The procedure involves the complete isotopic labeling of blue green algae (Spirulina platensis) with (15)N and utilizing it as a source of dietary nitrogen for mice. We outline a detailed protocol for in-house production of (15)N-labeled algae, labeling of mice, and analysis of isotope incorporation kinetics by mass spectrometry. The methodology can be adapted to analyze proteome dynamics in most murine tissues and may be particularly useful in the analysis of proteostatic disruptions in mouse models of disease. PMID:24791984

  3. 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.

  4. The Effect of N Fertilizer Placement on the Fate of Urea-15N and Yield of Winter Wheat in Southeast China

    PubMed Central

    Chen, Zhaoming; Wang, Huoyan; Liu, Xiaowei; Liu, Yongzhe; Gao, Shuaishuai; Zhou, Jianmin

    2016-01-01

    A field micro-plot experiment using nitrogen isotope (15N) labeling was conducted to determine the effects of placement methods (broadcast and band) and N rates (60, 150 and 240 kg ha–1) on the fate of urea-15N in the wheat–soil system in Guangde County of Anhui Province, China. N fertilizer applied in bands increased grain yield by 15% compared with broadcast application. The N fertilizer application rate had a significant effect on grain yield, straw yield and aboveground biomass, as well as on N uptake and N concentration of wheat. The recovery of urea-15N was a little higher for broadcast (34.0–39.0%) than for band treatment (31.2–38.2%). Most of the soil residual N was retained in the 0–20 cm soil layer. At the N rates of 60 and 240 kg ha–1, the residual 15N was higher for band (34.4 and 108.7 kg ha–1, respectively) than for broadcast application (29.6 and 88.4 kg ha–1, respectively). Compared with broadcast treatment, banded placement of N fertilizer decreased the N loss in the wheat–soil system. Band application one time is an alternative N management practice for winter wheat in this region. PMID:27082246

  5. Tracing the Cycling and Fate of the Explosive 2,4,6-Trinitrotoluene in Coastal Marine Systems with a Stable Isotopic Tracer, (15)N-[TNT].

    PubMed

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

    2015-10-20

    2,4,6-Trinitrotoluene (TNT) has been used as a military explosive for over a hundred years. Contamination concerns have arisen as a result of manufacturing and use on a large scale; however, despite decades of work addressing TNT contamination in the environment, its fate in marine ecosystems is not fully resolved. Here we examine the cycling and fate of TNT in the coastal marine systems by spiking a marine mesocosm containing seawater, sediments, and macrobiota with isotopically labeled TNT ((15)N-[TNT]), simultaneously monitoring removal, transformation, mineralization, sorption, and biological uptake over a period of 16 days. TNT degradation was rapid, and we observed accumulation of reduced transformation products dissolved in the water column and in pore waters, sorbed to sediments and suspended particulate matter (SPM), and in the tissues of macrobiota. Bulk δ(15)N analysis of sediments, SPM, and tissues revealed large quantities of (15)N beyond that accounted for in identifiable derivatives. TNT-derived N was also found in the dissolved inorganic N (DIN) pool. Using multivariate statistical analysis and a (15)N mass balance approach, we identify the major transformation pathways of TNT, including the deamination of reduced TNT derivatives, potentially promoted by sorption to SPM and oxic surface sediments. PMID:26375037

  6. (1)H, (13)C and (15)N backbone assignment of the EC-1 domain of human E-cadherin.

    PubMed

    Prasasty, Vivitri D; Krause, Mary E; Tambunan, Usman S F; Anbanandam, Asokan; Laurence, Jennifer S; Siahaan, Teruna J

    2015-04-01

    The Extracellular 1 (EC1) domain of E-cadherin has been shown to be important for cadherin-cadherin homophilic interactions. Cadherins are responsible for calcium-mediated cell-cell adhesion located at the adherens junction of the biological barriers (i.e., intestinal mucosa and the blood-brain barrier (BBB)). Cadherin peptides can modulate cadherin interactions to improve drug delivery through the BBB. However, the mechanism of modulating the E-cadherin interactions by cadherin peptides has not been fully elucidated. To provide a basis for subsequent examination of the structure and peptide-binding properties of the EC1 domain of human E-cadherin using solution NMR spectroscopy, the (1)H, (13)C and (15)N backbone resonance of the uniformly labeled-EC1 were assigned and the secondary structure was determined based on the chemical shift values. These resonance assignments are essential for assessing protein-ligand interactions and are reported here. PMID:24510398

  7. Comparison of the metabolism of [1,2,6,7-3H(N)]cholesteryl oleate, cholesteryl [9,10-3H]oleate, and cholesteryl [1-14C]oleate labeled lipoproteins in the rat.

    PubMed

    Terpstra, A H

    1994-04-01

    The intravascular metabolism of sterol labeled [1,2,6,7-3H(N)]cholesteryl oleate and acyl labeled cholesteryl [9,10-3H]oleate and cholesteryl [1-14C]oleate was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA). In a first series of studies, the metabolism of sterol labeled [1,2,6,7-3H(N)]cholesteryl oleate and acyl labeled cholesteryl [1-14C]oleate was compared, and the two tracers had identical plasma clearance rates when incorporated into human low density lipoproteins (LDL). The 3H sterol labeled cholesteryl ester (CE), however, had a plasma clearance rate lower than the 14C acyl labeled CE when incorporated into rat alpha- and beta-migrating LDL and human or rat high density lipoproteins (HDL). Unesterified 3H cholesterol reappeared in the plasma whereas the 14C radioactivity in the plasma remained associated with the CE. In a second set of studies, LDL and HDL were radiolabeled with cholesteryl [9,10-3H]oleate and cholesteryl [1-14C]oleate. Large amounts of 3H radioactivity that were dialyzable and not associated with the lipoprotein CE reappeared in the plasma during the kinetic studies. The two tracers had identical plasma disappearance rates when the plasma samples were dialyzed. The results of these studies indicate that the nature of the tracer used to trace lipoprotein CE can affect the estimated kinetic parameters of plasma CE. PMID:8060380

  8. Total-body protein turnover in parenterally fed neonates: effects of energy source studied by using [15N]glycine and [1-13C]leucine.

    PubMed

    Pencharz, P; Beesley, J; Sauer, P; Van Aerde, J; Canagarayar, U; Renner, J; McVey, M; Wesson, D; Swyer, P

    1989-12-01

    The effects of nonprotein energy source (ie, glucose only vs glucose and lipid) on nitrogen retention and total-body protein turnover were studied in 20 parenterally fed newborn infants. All infants received approximately 3 g amino acids and 80-90 kcal.kg body wt.d. Total-body protein synthesis was estimated by using three constant-infusion, end-product methods: enrichment of urinary urea and ammonia in response to a [15N]glycine label and exhaled carbon dioxide enrichment in response to a [1-13C]leucine label. No differences were seen in nitrogen retention between the two energy sources. The estimate of total-body protein turnover obtained from the 13C label was similar to that obtained with the [15N]urea label. No differences in turnover rates were observed between the two diet groups. Use of the glucose-plus-lipid fuel system enhanced energy storage and the reutilization of amino acid for protein synthesis. PMID:2512806

  9. Metabolism of mineral-sorbed organic matter and microbial lifestyles in fluvial ecosystems

    NASA Astrophysics Data System (ADS)

    Hunter, William Ross; Niederdorfer, Robert; Gernand, Anna; Veuger, Bart; Prommer, Judith; Mooshammer, Maria; Wanek, Wolfgang; Battin, Tom J.

    2016-02-01

    In fluvial ecosystems mineral erosion, carbon (C), and nitrogen (N) fluxes are linked via organomineral complexation, where dissolved organic molecules bind to mineral surfaces. Biofilms and suspended aggregates represent major aquatic microbial lifestyles whose relative importance changes predictably through fluvial networks. We tested how organomineral sorption affects aquatic microbial metabolism, using organomineral particles containing a mix of 13C, 15N-labeled amino acids. We traced 13C and 15N retention within biofilm and suspended aggregate biomass and its mineralization. Organomineral complexation restricted C and N retention within biofilms and aggregates and also their mineralization. This reduced the efficiency with which biofilms mineralize C and N by 30% and 6%. By contrast, organominerals reduced the C and N mineralization efficiency of suspended aggregates by 41% and 93%. Our findings show how organomineral complexation affects microbial C:N stoichiometry, potentially altering the biogeochemical fate of C and N within fluvial ecosystems.

  10. Indirect Measurement of {sup 15}N(p,{alpha}){sup 12}C and {sup 18}O(p,{alpha}){sup 15}N. Applications to the AGB Star Nucleosynthesis

    SciTech Connect

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

    2008-04-06

    The Trojan Horse Method has been recently applied to the study of reactions involved in fluorine nucleosynthesis inside AGB stars. Fluorine abundance is important since it allows to constrain mixing models from the comparison of the observed fluorine abundances with the ones predicted by models. Anyway direct measurements of the cross section do not extend down to the Gamow peak, which is the astrophysically relevant energy region. In particular the study focuses on the {sup 15}N(p,{alpha}){sup 12}C and the {sup 18}O(p,{alpha}){sup 15}N reactions which can influence fluorine yield as they are part of {sup 19}F production/destruction network.

  11. 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

  12. Climate-Dependence of Plant-Soil 15N/14N Interactions Across Tropical Rainforests

    NASA Astrophysics Data System (ADS)

    Houlton, B. Z.; Sigman, D. M.; Hedin, L. O.

    2005-12-01

    In most areas of the world, the 15N/14N of bulk soils is higher than that of plant leaves, and the isotopic signatures of these two ecosystem N pools progressively diverge with increasing rainfall. However, both the cause for this isotopic trend and its implications for understanding interactions between climate and N cycles are largely unknown. We report 15N/14N measurements of nitrate, ammonium, and total dissolved N in soil extracts from a highly constrained rainfall sequence in Hawaii, across which this trend in ecosystem 15N/14N is captured, to examine the competing explanations for plant-soil 15N/14N uncouplings. While the isotopic influences of microbial transfers of N between nitrate and ammonium pools and plant-mycorrhizae interactions have been posited in plant-soil 15N/14N relationships, our data did not support an important role for either of these mechanisms. Instead, preferential regeneration of 14N during the breakdown of DON to ammonium explains why the 15N/14N of plants is lower than that of bulk soils. Fractionation at this step leads to two isotopically distinct N subcycles in each forest, a lower-15N/14N subcycle composed of ammonium, nitrate, and bulk plant biomass N that `spins' rapidly and a higher-15N/14N subcycle composed of bulk soil N and DON that is much less dynamic. The increased difference between soil and plant 15N/14N is due to changes in the impacts of nitrification and denitrification on the 15N/14N of ammonium and nitrate, coupled with a switch from nitrate to ammonium uptake by plants under the wettest conditions. For instance, the particularly large (~6 per mil) 15N/14N difference between plants and soils in the wettest sites is due to the lack of 15N-enrichment of ammonium by nitrification coupled with plant dependence on ammonium uptake only. Our results highlight the importance of interactions between DON breakdown, ecosystem N recycling, and gaseous N losses in the explaining the interactions between the 15N signatures of

  13. Leaf δ(15)N as a physiological indicator of the responsiveness of N2-fixing alfalfa plants to elevated [CO2], temperature and low water availability.

    PubMed

    Ariz, Idoia; Cruz, Cristina; Neves, Tomé; Irigoyen, Juan J; Garcia-Olaverri, Carmen; Nogués, Salvador; Aparicio-Tejo, Pedro M; Aranjuelo, Iker

    2015-01-01

    The natural (15)N/(14)N isotope composition (δ(15)N) of a tissue is a consequence of its N source and N physiological mechanisms in response to the environment. It could potentially be used as a tracer of N metabolism in plants under changing environmental conditions, where primary N metabolism may be complex, and losses and gains of N fluctuate over time. In order to test the utility of δ(15)N as an indicator of plant N status in N2-fixing plants grown under various environmental conditions, alfalfa (Medicago sativa L.) plants were subjected to distinct conditions of [CO2] (400 vs. 700 μmol mol(-1)), temperature (ambient vs. ambient +4°C) and water availability (fully watered vs. water deficiency-WD). As expected, increased [CO2] and temperature stimulated photosynthetic rates and plant growth, whereas these parameters were negatively affected by WD. The determination of δ(15)N in leaves, stems, roots, and nodules showed that leaves were the most representative organs of the plant response to increased [CO2] and WD. Depletion of heavier N isotopes in plants grown under higher [CO2] and WD conditions reflected decreased transpiration rates, but could also be related to a higher N demand in leaves, as suggested by the decreased leaf N and total soluble protein (TSP) contents detected at 700 μmol mol(-1) [CO2] and WD conditions. In summary, leaf δ(15)N provides relevant information integrating parameters which condition plant responsiveness (e.g., photosynthesis, TSP, N demand, and water transpiration) to environmental conditions. PMID:26322051

  14. Leaf δ15N as a physiological indicator of the responsiveness of N2-fixing alfalfa plants to elevated [CO2], temperature and low water availability

    PubMed Central

    Ariz, Idoia; Cruz, Cristina; Neves, Tomé; Irigoyen, Juan J.; Garcia-Olaverri, Carmen; Nogués, Salvador; Aparicio-Tejo, Pedro M.; Aranjuelo, Iker

    2015-01-01

    The natural 15N/14N isotope composition (δ15N) of a tissue is a consequence of its N source and N physiological mechanisms in response to the environment. It could potentially be used as a tracer of N metabolism in plants under changing environmental conditions, where primary N metabolism may be complex, and losses and gains of N fluctuate over time. In order to test the utility of δ15N as an indicator of plant N status in N2-fixing plants grown under various environmental conditions, alfalfa (Medicago sativa L.) plants were subjected to distinct conditions of [CO2] (400 vs. 700 μmol mol−1), temperature (ambient vs. ambient +4°C) and water availability (fully watered vs. water deficiency—WD). As expected, increased [CO2] and temperature stimulated photosynthetic rates and plant growth, whereas these parameters were negatively affected by WD. The determination of δ15N in leaves, stems, roots, and nodules showed that leaves were the most representative organs of the plant response to increased [CO2] and WD. Depletion of heavier N isotopes in plants grown under higher [CO2] and WD conditions reflected decreased transpiration rates, but could also be related to a higher N demand in leaves, as suggested by the decreased leaf N and total soluble protein (TSP) contents detected at 700 μmol mol−1 [CO2] and WD conditions. In summary, leaf δ15N provides relevant information integrating parameters which condition plant responsiveness (e.g., photosynthesis, TSP, N demand, and water transpiration) to environmental conditions. PMID:26322051

  15. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

    PubMed Central

    Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

    2015-01-01

    To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources. PMID:26308446

  16. 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. PMID:25621737

  17. Production of 15N-depleted biomass during cyanobacterial N2-fixation at high Fe concentrations

    NASA Astrophysics Data System (ADS)

    Zerkle, Aubrey L.; Junium, Christopher K.; Canfield, Donald E.; House, Christopher H.

    2008-09-01

    In this study we examine the effects of varying Fe, Mo, and P concentrations on δ15N fractionation during N2 fixation in the cyanobacterium Anabaena variabilis. We show that when grown in Fe-enriched media ([Fe] ≥ 50 nM), this organism produces biomass up to 3‰ lower in δ15N than when grown in Fe-limited media ([Fe] < 50 nM). A compilation of our data with previous measurements of δ15N in N2-fixing cyanobacteria reveals a general trend toward the production of more 15N-depleted biomass at higher Fe concentrations. We discuss our results in the context of negative δ15N values preserved in Archean and some Phanerozoic sediments, generally attributed to the production of marine organic matter with low δ15N by N2 fixation (and potentially NH4+ regeneration) during periods of fluctuating nutrient dynamics. We suggest that enhanced Fe availability during periods of widespread ocean anoxia can further stimulate the production of 15N-depleted biomass by N2-fixing organisms, contributing to the isotopic record.

  18. The products of the reduction of doxyl stearates in cells are hydroxylamines as shown by oxidation by 15N-perdeuterated Tempone.

    PubMed

    Chen, K; Swartz, H M

    1989-07-21

    The use of nitroxides in functional biological systems has increased greatly as it has become evident that such studies can provide valuable biophysical and metabolic data. This has led to a need to understand the nature of the metabolism of nitroxides and their products. This paper presents data indicating the value of 15N-perdeuterated Tempone specifically to indicate the amount of hydroxylamines that are present in a cellular system. Using this technique, we found that in the mammalian cells that we studied the principal or only products of reduction of doxyl stearates were the corresponding hydroxylamines. PMID:2546601

  19. Radiative p 15N Capture in the Region of Astrophysical Energies

    NASA Astrophysics Data System (ADS)

    Dubovichenko, S. B.; Burtebaev, N.; Dzhazairov-Kakhramanov, A. V.; Alimov, D. K.

    2016-06-01

    Within the framework of the modified potential cluster model with classification of orbital states according to the Young schemes, the possibility of describing experimental data for the astrophysical S-factor of p 15N radiative capture at energies from 50 to 1500 keV is considered. It is shown that on the basis of M1 and E1 transitions from various p 15N scattering states to the ground state of the 16O nucleus in the p 15N channel it is entirely possible to successfully explain the overall behavior of the S-factor in the considered energy region in the presence of two resonances.

  20. Nitrogen dynamics in a Western Boundary Upwelling System (Cabo Frio, Brazil) based on δ15N-nitrate and δ15N of sinking particle signals

    NASA Astrophysics Data System (ADS)

    Fontana, L.; Belem, A. L.; Venancio, I.; Duarte, C.; Chiara, S. D.; Albuquerque, A. L.

    2014-12-01

    To improve the efficiency of upwelling to control nitrogen dynamic in the ocean, better understanding of the occurring processes is necessary. This research explores δ15N of nitrate and sinking particles on a western boundary upwelling System (Cabo Frio, Brazil). The Continental Shelf of southeastern Brazil is dominated by the oligotrophic Brazil Current, whose instabilities promote the coastal upwelling of South Atlantic Central Water (SACW), and consequently increases of primary productivity. The coastal upwelling system plays an important role in the nitrogen dynamics on the Cabo Frio Upwelling System (CFUS). However, the interactions between biological induced processes, including biological N-fixation and nitrate inputs from upwelled waters in CFUS still have not been well explored. Then, this study aims clarify N-dynamics on CFUS based on a cross-shelf approach. δ15N-nitrate was characterized for each water masses present on the shelf (South Atlantic Central Water, Tropical Water and Coastal Water) and associated with physicochemical parameters (T/S, nutrients), as well as the δ15N of sinking particles at different depths (from surface to the bottom water). Samples were collected in a time interval of 1 month during ~4 years (2011 to 2014). Cross-shelf gradients of nitrogen species concentration (ammonium + nitrite + nitrate) and stable isotopes were observed. The δ15N of nitrate and sinking particles were interpreted according to the prevailing processes of the N-transformations. Considering the region as N-limited (N:P < 16), processes as biological N-fixation seems to be dominant on oligotrophic Tropical Waters. Coastal upwelled SACW showed δ15N-nitrate signature within the global average of deep ocean (5-6‰) characterizing the inner and mid-shelf conditions, where the input of new nitrate from upwelling is rapidly used by organisms in the euphotic zone without any fractionation. On the other hands, the dominance of N-limited Tropical Waters on the

  1. Carbon-rich Presolar Grains from Massive Stars: Subsolar 12C/13C and 14N/15N Ratios and the Mystery of 15N

    NASA Astrophysics Data System (ADS)

    Pignatari, M.; Zinner, E.; Hoppe, P.; Jordan, C. J.; Gibson, B. K.; Trappitsch, R.; Herwig, F.; Fryer, C.; Hirschi, R.; Timmes, F. X.

    2015-08-01

    Carbon-rich grains with isotopic anomalies compared to the Sun are found in primitive meteorites. They were made by stars, and carry the original stellar nucleosynthesis signature. Silicon carbide grains of Type X and C and low-density (LD) graphites condensed in the ejecta of core-collapse supernovae. We present a new set of models for the explosive He shell and compare them with the grains showing 12C/13C and 14N/15N ratios lower than solar. In the stellar progenitor H was ingested into the He shell and not fully destroyed before the explosion. Different explosion energies and H concentrations are considered. If the supernova shock hits the He-shell region with some H still present, the models can reproduce the C and N isotopic signatures in C-rich grains. Hot-CNO cycle isotopic signatures are obtained, including a large production of 13C and 15N. The short-lived radionuclides 22Na and 26Al are increased by orders of magnitude. The production of radiogenic 22Ne from the decay of 22Na in the He shell might solve the puzzle of the Ne-E(L) component in LD graphite grains. This scenario is attractive for the SiC grains of type AB with 14N/15N ratios lower than solar, and provides an alternative solution for SiC grains originally classified as nova grains. Finally, this process may contribute to the production of 14N and 15N in the Galaxy, helping to produce the 14N/15N ratio in the solar system.

  2. Solution 1H, 15N NMR spectroscopic characterization of substrate-bound, cyanide-inhibited human heme oxygenase: water occupation of the distal cavity.

    PubMed

    Li, Yiming; Syvitski, Ray T; Auclair, Karine; Ortiz de Montellano, Paul; La Mar, Gerd N

    2003-11-01

    A solution NMR spectroscopic study of the cyanide-inhibited, substrate-bound complex of uniformly (15)N-labeled human heme oxygenase, hHO, has led to characterization of the active site with respect to the nature and identity of strong hydrogen bonds and the occupation of ordered water molecules within both the hydrogen bonding network and an aromatic cluster on the distal side. [(1)H-(15)N]-HSQC spectra confirm the functionalities of several key donors in particularly robust H-bonds, and [(1)H-(15)N]HSQC-NOESY spectra lead to the identification of three additional robust H-bonds, as well as the detection of two more relatively strong H-bonds whose identities could not be established. The 3D NMR experiments provided only a modest, but important, extension of assignments because of the loss of key TOCSY cross-peaks due to the line broadening from a dynamic heterogeneity in the active site. Steady-state NOEs upon saturating the water signal locate nine ordered water molecules in the immediate vicinity of the H-bond donors, six of which are readily identified in the crystal structure. The additional three are positioned in available spaces to account for the observed NOEs. (15)N-filtered steady-state NOEs upon saturating the water resonances and (15)N-filtered NOESY spectra demonstrate significant negative NOEs between water molecules and the protons of five aromatic rings. Many of the NOEs can be rationalized by water molecules located in the crystal structure, but strong water NOEs, particularly to the rings of Phe47 and Trp96, demand the presence of at least an additional two immobilized water molecules near these rings. The H-bond network appears to function to order water molecules to provide stabilization for the hydroperoxy intermediate and to serve as a conduit to the active site for the nine protons required per HO turnover. PMID:14583035

  3. Increase of Natural 15N Enrichment of Soybean Nodules with Mean Nodule Mass 1

    PubMed Central

    Shearer, Georgia; Bryan, Barbara A.; Kohl, Daniel H.

    1984-01-01

    The 15N abundance of soybean (Glycine max L. Merrill var Harosoy) nodules is usually greater than it is for other tissues or for atmospheric N2. Results of experiments in which nodules were separated by size show that the magnitude of the 15N enrichment is correlated with nodule mass. The results support the hypothesis that 15N enrichment of nodules results from differential N isotopic fractionation for synthesis of nodule tissue versus synthesis of compounds for export from the nodule. The physiological significance of this hypothesis is that it requires that a substantial fraction of the N for nodule tissue synthesis in 15N-enriched nodules be N recently fixed within the same nodule. PMID:16663917

  4. 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

  5. Sources of δ15N variability in sinking particulate nitrogen in the Cariaco Basin, Venezuela

    NASA Astrophysics Data System (ADS)

    Montes, Enrique; Thunell, Robert; Muller-Karger, Frank E.; Lorenzoni, Laura; Tappa, Eric; Troccoli, Luis; Astor, Yrene; Varela, Ramón

    2013-09-01

    Ten years of monthly observations of the δ15N of sinking particulate nitrogen (δ15N-PN (in ‰ versus atmospheric N2)=[(15N/14N)sample/(15N/14N)standard)-1]1000) in the Cariaco Basin, Venezuela, confirm that the basin's bottom sediments store information about nitrogen dynamics related to seasonal and interannual variability in regional surface ocean processes. During the upwelling period of the southern Caribbean Sea (February-April), the δ15N-PN is similar to that of the thermocline nitrate (˜3.5‰). This nitrate is imported into the Cariaco Basin with Subtropical Underwater (SUW), which wells up near the coast. Thus, particles generated by phytoplankton photosynthesis during this productive period bear a sub-tropical North Atlantic isotopic imprint of N2 fixation (low compared to the global average of nitrate δ15N≈5‰). During the non-upwelling period when surface waters are stratified (September-November), the δ15N-PN is also 3.5-4.0‰, and reflects a mixture of local N2 fixation within the mixed layer, inputs of terrigenous organic matter and SUW nitrate consumption by phytoplankton below the mixed layer, which most likely exerts the strongest control on the δ15N-PN signal during this time. In the transition periods of May-July and December-January, the δ15N-PN increases to 4.5-6.5‰. This coincides with maxima of continental material fluxes (terrestrial PON δ15N is >6‰) into the Cariaco Basin. The δ15N signal in the sediments of the Cariaco Basin thus provides information about the relative strength of the local coastal upwelling, the relative input of continental material via river runoff, and local N2 fixation. The findings contribute to interpretations of the basin's paleoclimatic nitrogen cycle variations based on observations of the sedimentary δ15N record at this location.

  6. 15N fractionation in star-forming regions and Solar System objects

    NASA Astrophysics Data System (ADS)

    Wirström, Eva; Milam, Stefanie; Adande, Gilles; Charnley, Steven B.; Cordiner, Martin A.

    2015-08-01

    A central issue for understanding the formation and evolution of matter in the early Solar System is the relationship between the chemical composition of star-forming interstellar clouds and that of primitive Solar System materials. The pristine molecular content of comets, interplanetary dust particles and carbonaceous chondrites show significant bulk nitrogen isotopic fractionation relative to the solar value, 14N/15N ~ 440. In addition, high spatial resolution measurements in primitive materials locally show even more extreme enhancements of 14N/15N < 100.The coherent 15N enrichment in comets from different formation zones suggests that these isotopic enhancements are remnants of the interstellar chemistry in the natal molecular cloud core and the outer protosolar nebula. Indeed, early chemical models of gas-phase ion-molecule nitrogen fractionation showed that HCN and HNC (nitriles) can hold significant 15N enrichments in cold dark clouds where CO is depleted onto dust grains. In addition, 15N fractionation in nitriles and amines (NH2, NH3) follow different chemical pathways. More recently we have shown that once the spin-state dependence in rates of reactions with H2 is included in the models, amines can either be enhanced or depleted in 15N, depending on the core’s evolutionary stage. Observed 15N fractionation in amines and nitriles therefore cannot be expected to be the same, instead their ratio is a potential chemical clock.Observations of molecular isotope ratios in dark cores are challenging. Limited published results in general show higher 15N/14N ratios in HCN and HNC than ammonia, but more measurements are necessary to confirm these trends. We will present recent results from our ongoing observing campaign of 14N/15N isotopic ratios in HCN, HNC and NH3 in dense cores and protostars which seem consistent with significant fractionation in nitriles as compared to other molecules in each object. The few 14N/15N ratios observed in N2H+ are similar to

  7. Water proton spin saturation affects measured protein backbone 15 N spin relaxation rates

    NASA Astrophysics Data System (ADS)

    Chen, Kang; Tjandra, Nico

    2011-12-01

    Protein backbone 15N NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses. Here different water suppression methods were incorporated into pulse sequences to measure 15N longitudinal T1 and transversal rotating-frame T1ρ spin relaxation. Unexpectedly the 15N T1 relaxation time constants varied significantly with the choice of water suppression method. For a 25-kDa Escherichiacoli. glutamine binding protein (GlnBP) the T1 values acquired with the pulse sequence containing a water dephasing gradient are on average 20% longer than the ones obtained using a pulse sequence containing the water flip-back pulse. In contrast the two T1ρ data sets are correlated without an apparent offset. The average T1 difference was reduced to 12% when the experimental recycle delay was doubled, while the average T1 values from the flip-back measurements were nearly unchanged. Analysis of spectral signal to noise ratios ( s/ n) showed the apparent slower 15N relaxation obtained with the water dephasing experiment originated from the differences in 1H N recovery for each relaxation time point. This in turn offset signal reduction from 15N relaxation decay. The artifact becomes noticeable when the measured 15N relaxation time constant is comparable to recycle delay, e.g., the 15N T1 of medium to large proteins. The 15N relaxation rates measured with either water suppression schemes yield reasonable fits to the structure. However, data from the saturated scheme results in significantly lower Model-Free order parameters (< S2> = 0.81) than the non-saturated ones (< S2> = 0.88), indicating such order parameters may be previously underestimated.

  8. 15N techniques and analytical procedures. Indo/U. S. science and technology initiative. Research report

    SciTech Connect

    Porter, L.K.; Mosier, A.R.

    1992-05-01

    (15)N technology is used to explore many agricultural research topics, including the movement of nitrates to groundwater, use of fertilizer nitrogen by plants, ways to increase nitrogen fixation, and effects of management practices on denitrification. The publication reviews (15)N procedures and methods for handling and collecting samples, introducing isotopes into plants and soils, and for performing Kjeldahl analyses, isotope dilutions, Rittenberg oxidation conversions for isotope-ration analyses, and automated Dumas isotope-ratio analyses.

  9. 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.

  10. Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids.

    PubMed

    Jordà, Joel; de Jesus, Sérgio S; Peltier, Solenne; Ferrer, Pau; Albiol, Joan

    2014-01-25

    The yeast Pichia pastoris has emerged as one of the most promising yeast cell factories for the production of heterologous proteins. The readily available genetic tools and the ease of high-cell density cultivations using methanol or glycerol/methanol mixtures are among the key factors for this development. Previous studies have shown that the use of mixed feeds of glycerol and methanol seem to alleviate the metabolic burden derived from protein production, allowing for higher specific and volumetric process productivities. However, initial studies of glycerol/methanol co-metabolism in P. pastoris by classical metabolic flux analyses using (13)C-derived Metabolic Flux Ratio (METAFoR) constraints were hampered by the reduced labelling information obtained when using C3:C1 substrate mixtures in relation to the conventional C6 substrate, that is, glucose. In this study, carbon flux distributions through the central metabolic pathways in glycerol/methanol co-assimilation conditions have been further characterised using biosynthetically directed fractional (13)C labelling. In particular, metabolic flux distributions were obtained under 3 different glycerol/methanol ratios and growth rates by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids using the software tool (13)CFlux2. Specifically, cells were grown aerobically in chemostat cultures fed with 80:20, 60:40 and 40:60 (w:w) glycerol/methanol mixtures at two dilutions rates (0.05 hour(-1) and 0.16 hour(-1)), allowing to obtain additional data (biomass composition and extracellular fluxes) to complement pre-existing datasets. The performed (13)C-MFA reveals a significant redistribution of carbon fluxes in the central carbon metabolism as a result of the shift in the dilution rate, while the ratio of carbon sources has a lower impact on carbon flux distribution in cells growing at the same dilution rate. At low growth rate, the percentage of methanol directly dissimilated to CO2 ranges

  11. Elastic and inelastic scattering of 15N ions by 9Be at 84 MeV

    NASA Astrophysics Data System (ADS)

    Rudchik, A. T.; Chercas, K. A.; Kemper, K. W.; Rusek, K.; Rudchik, A. A.; Herashchenko, O. V.; Koshchy, E. I.; Pirnak, Val. M.; Piasecki, E.; Trzcińska, A.; Sakuta, S. B.; Siudak, R.; Strojek, I.; Stolarz, A.; Ilyin, A. P.; Ponkratenko, O. A.; Stepanenko, Yu. M.; Shyrma, Yu. O.; Szczurek, A.; Uleshchenko, V. V.

    2016-03-01

    Angular distributions of the 9Be + 15N elastic and inelastic scattering were measured at Elab(15N) = 84 MeV (Ec.m. = 31.5 MeV) for the 0-6.76 MeV states of 9Be and 0-6.32 MeV states of 15N. The data were analyzed within the optical model and coupled-reaction-channels method. The elastic and inelastic scattering, spin reorientations of 9Be in ground and excited states and 15N in excited states as well as the most important one- and two-step transfer reactions were included in the channels-coupling scheme. The parameters of the 9Be + 15N optical potential of Woods-Saxon form as well as deformation parameters of these nuclei were deduced. The analysis showed that the 9Be + 15N pure potential elastic scattering dominates at the forward angles whereas the ground state spin reorientation of 9Be gives a major contribution to the elastic scattering cross sections at the large angles. Contributions from particle transfers are found to be negligible for the present scattering system.

  12. Highly 15N-Enriched Chondritic Clasts in the Isheyevo Meteorite

    SciTech Connect

    Bonal, L; Huss, G R; Krot, A N; Nagashima, K; Ishii, H A; Bradley, J P; Hutcheon, I D

    2009-01-14

    The metal-rich carbonaceous chondrites (CB and CH) have the highest whole-rock {sup 15}N enrichment ({delta}{sup 15}N up to +1500{per_thousand}), similar to {delta}{sup 15}N values reported in micron-sized regions (hotspots) of Interplanetary Dust Particles (IDPs) of possibly cometary origin and fine-grained matrices of unmetamorphosed chondrites. These {sup 15}N-rich hotspots are commonly attributed to low-temperature ion-molecule reactions in the protosolar molecular cloud or in the outer part of the protoplanetary disk. The nature of the whole-rock {sup 15}N enrichment of the metal-rich chondrites is not understood. We report a discovery of a unique type of primitive chondritic clasts in the CH/CB-like meteorite Isheyevo, which provides important constraints on the origin of {sup 15}N anomaly in metal-rich chondrites and nitrogen-isotope fractionation in the Solar System. These clasts contain tiny chondrules and refractory inclusions (5-15 {micro}m in size), and abundant ferromagnesian chondrule fragments (1-50 {micro}m in size) embedded in the partly hydrated, fine-grained matrix material composed of olivines, pyroxenes, poorly-organized aromatic organics, phyllosilicates and other hydrous phases. The mineralogy and oxygen isotope compositions of chondrules and refractory inclusions in the clasts are similar to those in the Isheyevo host, suggesting formation at similar heliocentric distances. In contrast to the previously known extraterrestrial samples, the fine-grained material in the clasts is highly and rather uniformly enriched in {sup 15}N, with bulk {delta}{sup 15}N values ranging between +1000 and +1300{per_thousand}; the {delta}{sup 15}N values in rare hotspots range from +1400 to +4000{per_thousand}. Since fine-grained matrices in the lithic clasts are the only component containing thermally unprocessed (during CAI and chondrule formation or during impact melting) materials that accreted into the metal rich chondrite parent body(ies), the {sup 15}N

  13. Metabolic Changes Associated with Adaptation of Plant Cells to Water Stress 1

    PubMed Central

    Rhodes, David; Handa, Sangita; Bressan, Ray A.

    1986-01-01

    Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from 15N labeling kinetics after substituting [15N]ammonium and [15N]nitrate for the 14N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and γ-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and

  14. An open-label, single-dose, crossover study of the pharmacokinetics and metabolism of two oral formulations of 1-octanol in patients with essential tremor.

    PubMed

    Nahab, Fatta B; Wittevrongel, Loretta; Ippolito, Dominic; Toro, Camilo; Grimes, George J; Starling, Judith; Potti, Gopal; Haubenberger, Dietrich; Bowen, Daniel; Buchwald, Peter; Dong, Chuanhui; Kalowitz, Daniel; Hallett, Mark

    2011-10-01

    Existing therapeutic options for management of essential tremor are frequently limited by poor efficacy and adverse effects. Likely the most potent tremor suppressant used is ethanol, although its use is prohibitive due to a brief therapeutic window, and the obvious implications of excessive alcohol use. Longer-chain alcohols have been shown to suppress tremor in harmaline animal models, and appear to be safe and well tolerated in 2 prior studies in humans. Here we report on the findings of a phase I/II study of 1-octanol designed to explore pharmacokinetics, efficacy, and safety. The most significant finding was the identification of octanoic acid as the product of rapid 1-octanol metabolism. Furthermore, the temporal profile of efficacy closely matches the plasma concentration of octanoic acid. Therefore, these findings identify a novel class of compound (e.g., carboxylic acids) with tremor suppressive properties in ET. Administration of 1-octanol also appears to be safe based on various measures collected. Essential tremor (ET) is the most common tremor disorder, with tremors occurring during static posturing or movement. These tremors are known to briefly improve in many cases after alcohol (ethanol) consumption. Two previous studies of a longer chain alcohol, 1-octanol, have demonstrated longer duration tremor-suppressive effects without the occurrence of intoxication. The aim of this study was to characterize the pharmacokinetics of 1-octanol and its primary metabolite octanoic acid using two formulations, along with additional safety and efficacy measures. Participants with proven ethanol-responsive ET were recruited into 1 of 2 parts: (part A) a dose escalation study (1-64 mg/kg; n = 4), and (part B) a fixed dose (64 mg/kg; n = 10) balanced, open-label crossover design. Two participants in part B then completed an exploratory part C evaluating 128 mg/kg.Plasma samples were collected at 10 intervals during a 6-hour period postingestion. Efficacy was

  15. 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.

  16. Sewage derive [sup 15]N in the Baltic traced in fucus

    SciTech Connect

    Hobbie, J.E.; Fry, B. ); Larsson, U.; Elmgren, R. )

    1990-01-09

    Himmerfjarden, a fjord-like bay on the eastern shore of the Baltic, receives treated sewage from 250,000 inhabitants. Because the inorganic N in the effluent is enriched in [sup 15]N through denitrification, nitrification, and ammonia volatilization, an analysis of the distribution of [sup 15]N in the Bay tells how far from the source the sewage nitrogen moves. The attached macroalga Fucus vesiculosus was collected in early May from rocky shore at 0-0.5 m depth and the [sup 15]N content of the tips of the fronds analyzed. This N represents uptake and storage during the previous six months and growth during March and April. The [delta][sup 15]N was uniformly high (11-13[per thousand]) in the main body of the Bay within 15 km from the sewage source. Beyond 15 km values decreased with distance to a low of 4.6[per thousand] at 35 km, where the Bay ends and the coastal waters begin. Using the 11-13 and 4.6[per thousand] as endmembers, the percentage of sewage N making up the Fucus at any point may be calculated. The [delta][sub 15]N of particulate organic matter in the offshore Baltic waters was around 0[per thousand] and Fucus had an [delta][sup 15]N about 1.5[per thousand] higher than the POM. From this and other evidence we conclude that there is a belt of coastal water with an elevated [delta][sup 15]N lying along the east coast of the Baltic. This presumably derives from sewage and perhaps from agriculture and is potentially of use as a tracer of coastal zone/pelagic zone interactions.

  17. Why is Mineral-Associated Organic Matter Enriched in 15N? Evidence from Grazed Pasture Soil

    NASA Astrophysics Data System (ADS)

    Baisden, W. T.; Wells, N. S.; Mudge, P. L.; Clough, T. J.; Schipper, L. A.; Ghani, A.; Stevenson, B.

    2014-12-01

    Throughout the scientific literature, measurements across soil depth and density fractions suggest that, with few exceptions, mineral-associated organic matter (OM) has higher δ15N than non-mineral-associated OM. This implies that the δ15N difference between N inputs and mineral-stabilized OM may characterize the microbial processes involved in stabilization and mineral association. Yet current understanding of observed N isotope fractionation in terrestrial ecosystems suggests the large isotope effects are expressed during inorganic N transformations from NH4 to gaseous loss pathways of NH3 volatilization and denitrification. How can the relative importance of N isotope fractionation during OM stabilization versus loss pathways be resolved? We recently examined N isofluxes when a temporary nitrogen excess is created by urine deposition in a New Zealand dairy pasture. We found that the N isotopic composition of volatilized NH3, and NO3 available for leaching or denitrification could not be linked back to the added N using Rayleigh distillation models. Instead, the results imply that the added N was immobilized, and the N available for losses was increasingly derived from mineralization of organic matter during the course of the experiment. These results are consistent with recent evidence of enhanced OM mineralization in urine patches, understanding of N isotope mass balances and long-standing evidence that gross mineralization and immobilization fluxes greatly exceed net mineralization and nitrification, except at very high N saturation. These results suggest that where 15N enrichment occurs due to fractionating loss pathways, the isotope effects are primarily transmitted to immobilized N, forming 15N enriched stabilized OM. This further explains earlier findings that the δ15N of soil OM represents an integrated indicator of losses, reflecting the intensity and duration of pastoral agriculture. We suggest that development of an indicator based on δ15N in

  18. 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. PMID:20048329

  19. 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

  20. 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

  1. 15N and 1H NMR spectroscopy of the catalytic histidine in chloromethyl ketone-inhibited complexes of serine proteases.

    PubMed

    Tsilikounas, E; Rao, T; Gutheil, W G; Bachovchin, W W

    1996-02-20

    The hemiketal hydroxyl groups in chloromethyl ketone (cmk) complexes of trypsin and chymotrypsin have been reported to ionize to the oxyanion with pK(a) values 2-4 pK(a) units below expectations for such a functional group on the basis of the behavior of the hemiketal carbon atom in 13C NMR spectra [Finucane, M. D., & Malthouse, J. P. G. (1992) Biochem. J. 286, 889-900]. The low pK(a) indicates the enzymes selectively stabilize the oxyanion form of the bound inhibitor, and therefore that cmk complexes may be good models of enzyme-mediated transition-state stabilization. However, the 13C NMR studies could not rule out His57 as the titrating group. Here we report the behavior of the ring 15N atoms of His57 in the Ala-Ala-Pro-Val-cmk complex of alpha-lytic protease. Both N(delta 1) and N(epsilon 2) of His57 respond to an ionization with a pK(a) of approximately 7.5, but His57 itself does not titrate as N(epsilon 2) remains alkylated and N(delta 1) remains bonded to a proton over the entire pH range. The species titrating with a pK(a) of approximately 7.5 must therefore be the hemiketal hydroxyl. The results also show that the 1H NMR signal from the proton in the Asp-His hydrogen bond behaves in a characteristic manner in cmk complexes and can be used diagnostically to confirm that His57 does not titrate and to measure the pK(a) of the hemiketal hydroxyl in cmk-protease complexes without resorting to 15N-labeling. We have used the behavior of this signal to directly confirm that His57 does not titrate in the trypsin and chymotrypsin complexes that were the subjects of the original 13C NMR studies. PMID:8652587

  2. 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

  3. Mechanistic Determination of Nitrogen Removal By Advanced Soil-Based Wastewater Treatment Systems Using 15n Isotopes

    NASA Astrophysics Data System (ADS)

    Cooper, J.; Loomis, G.; Kalen, D.; Boving, T. B.; Morales, I.; Amador, J.

    2014-12-01

    Current levels of nitrogen removal by onsite wastewater treatment systems (OWTS) are inadequate, with release of N from OWTS contributing to environmental N pollution, especially in coastal zones where aquatic ecosystems are sensitive to eutrophication. Current mechanistic understand of N removal are limited and mainly attributed to denitrification in the drainfield. Loss of N from N2O production during nitrification, a sparsely researched topic, may be a significant mechanism in advanced OWTS systems that enhance O2 diffusion by sand filter pre-treatment, shallow placement of infiltrative areas and timed dosing controls to prevent drainfield saturation. Replicate (n=3) intact soil mesocosms were used with 15N isotope to evaluate the effectiveness and mechanisms of N removal in drainfields with a conventional wastewater delivery (pipe-and-stone, P&S) compared to two advanced types of drainfields, pressurized shallow narrow drainfield (SND) and Geomat (GEO), a variation of a SND drainfield. Over the 11 day experiment, dissolved O2 was 1.6 mg/L for P&S and 3.0 mg/L for SND and GEO. Removal of total N was 13.5% for P&S, 4.8% for SND and 5.4% for GEO. 15NH4 labeled nitrogen inputs to drainfields were transformed primarily to 15NO3 in all outputs. Consistent low 15N2O levels were present in P&S, with increasing levels of N2 peaking 48h after 15NH4 injection, suggesting denitrification dominated N removal. By contrast, SND and GEO 15N2O levels rose quickly, peaking 8h after 15NH4 injection, suggesting N loss by nitrification. When the whole system is considered, including sand filter removal, 26 - 27% of total N was removed by the SND and GEO systems, whereas 14% of total N was removed in the P&S system. Our results suggest the SND and GEO systems as a whole are capable of removing a greater mass of N than the P&S system.

  4. Highly 15N-enriched chondritic clasts in the CB/CH-like meteorite Isheyevo

    NASA Astrophysics Data System (ADS)

    Bonal, L.; Huss, G. R.; Krot, A. N.; Nagashima, K.; Ishii, H. A.; Bradley, J. P.

    2010-11-01

    The metal-rich carbonaceous chondrites (CB and CH) have the highest whole-rock 15N-enrichments (δ 15N up to 1500‰) among planetary materials. They are also characterized by the absence of interchondrule fine-grained matrix. The only fine-grained material is present as lithic clasts, which experienced extensive aqueous alteration in contrast to the surrounding high-temperature components (chondrules, refractory inclusions, metal grains). Hence, the clasts are foreign objects that were incorporated at a late stage into the final parent body of Isheyevo. Their origin is poorly constrained. Based on mineralogy, petrography, and thermal processing of the aromatic carbonaceous component, different types of clasts have been previously identified in the CB/CH-like chondrite Isheyevo. Here, we focus on the rare lithic clasts characterized by the presence of anhydrous silicates (chondrules, chondrule fragments, and CAIs). Their mineralogy and oxygen isotopic compositions reveal them to be micro-chondrules, fragments of chondrules, and refractory inclusions related to those in the Isheyevo host, suggesting accretion in the same region. In contrast to previously studied IDPs or primitive chondritic matrices, the fine-grained material in the clasts we studied is highly and rather uniformly enriched in heavy nitrogen, with bulk δ 15N values ranging between 1000‰ and 1300‰. It is also characterized by the presence of numerous 15N hotspots (δ 15N ranging from 1400‰ to 4000‰). No bulk (δD <-240‰) or localized deuterium enrichments were observed. These clasts have the highest bulk enrichment in heavy nitrogen measured to date in a fine-grained material. They represent a unique material, of asteroidal or cometary origin, in our collection of cosmomaterials. We show that they were 15N-enriched before their incorporation in the final parent body of Isheyevo. They experienced an extensive aqueous alteration that most likely played a role in redistributing 15N over the

  5. Nitrogen Isotopic Ratios in Cometary NH2: Implication for 15N-fractionation in Ammonia

    NASA Astrophysics Data System (ADS)

    Shinnaka, Yoshiharu; Kawakita, Hideyo; Jehin, Emmanuël; Decock, Alice; Hutsemékers, Damien; Manfroid, Jean; Arai, Akira

    2015-11-01

    Isotopic ratios in cometary molecules are diagnostic for the physico-chemical conditions where molecules formed and are processed, from the interstellar medium to the solar nebula. Usually temperatures at the molecular formation control the fractionation of the heavier element in molecular species, e.g., D-fractionation in water.In cometary volatiles, the 14N/15N ratios in CN have been well observed (Manfroid et al. 2009, A&A, 503, 613, and reference therein) and is consistent with the ratio in HCN (a most probable parent of CN) measured in few comets (Bockelée-Morvan et al. 2008, ApJ, 679, L49). Those ratios are enriched compared to the proto-solar value by a factor of ~3. In contrast to those Nitriles, there are only few reports on 14N/15N ratios in Ammonia (as Amine) (Rousselot et al. 2014, ApJ, 780, L17; Shinnaka et al. 2014, ApJ, 782, L16). Ammonia (NH3) is usually the most abundant and HCN is the second most abundant N-bearing volatiles in cometary ice. Especially, recent observations of 15NH2 revealed the 14N/15N ratios in NH3 are comparable to those of CN. However, from the viewpoint of theoretical work, the enrichment of 15N in cometary NH3 cannot be reproduced by current chemical network models. Information about the diversity of the 14N/15N ratios in NH3 of individual comets is needed to understand the formation mechanisms/environments of NH3 in the early solar system.To clarify the diversity of the 14N/15N ratios in cometary NH3, we determine the 14N/15N ratios in NH3 for more than ten comets individually which include not only Oort cloud comets but also short period comets by using the high-resolution optical spectra of NH2. These spectra were obtained with both the UVES mounted on the VLT in Chile and the HDS on the Subaru Telescope in Hawaii.The derived 14N/15N ratios in NH3 for more than ten comets show high 15N-enrichment compared with the elemental abundances of nitrogen in the Sun by about factor of ~3 and has no large diversity depending on

  6. 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.

  7. Hydrogen Bonds in Crystalline Imidazoles Studied by 15N NMR and ab initio MO Calculations

    NASA Astrophysics Data System (ADS)

    Ueda, Takahiro; Nagatomo, Shigenori; Masui, Hirotsugu; Nakamura, Nobuo; Hayashi, Shigenobu

    1999-07-01

    Intermolecular hydrogen bonds of the type N-H...N in crystals of imidazole and its 4-substituted and 4,5-disubstituted derivatives were studied by 15N CP/MAS NMR and an ab initio molecular orbital (MO) calculation. In the 15N CP/MAS NMR spectrum of each of the imidazole derivatives, two peaks due to the two different functional groups, >NH and =N-, were observed. The value of the 15N isotropic chemical shift for each nitrogen atom depends on both the length of the intermolecular hydrogen bond and the kind of the substituent or substituents. It was found that the difference between the experimen-tal chemical shifts of >NH and =N-varies predominantly with the hydrogen bond length but does not show any systematic dependence on the kind of substituent. The ab initio MO calculations suggest that the hydrogen bond formation influences the 15N isotropic chemical shift predominantly, and that the difference between the 15N isotropic chemical shift of >NH and =N-varies linearly with the hydrogen bond length.

  8. Nitrogen recycling during phenylpropanoid metabolism in sweet potato tubers

    NASA Technical Reports Server (NTRS)

    Singh, S.; Lewis, N. G.; Towers, G. H.

    1998-01-01

    In the first step of the phenylpropanoid metabolic pathway, L-phenylalanine (L-Phe) is deaminated to form E-cinnamate, in a conversion catalyzed by phenylalanine ammonia-lyase (PAL; EC 4.3.1.5). The metabolic fate of the ammonium ion (NH4+) produced in this reaction was investigated in sweet potato (Ipomoea batatas) tuber discs. [15N]-Labeled substrates including L-Phe, in the presence or absence of specific enzyme inhibitors, were administered to sweet potato discs in light under aseptic conditions. 15N-Nuclear magnetic resonance spectroscopic analyses revealed that the 15NH4+ liberated during the PAL reaction is first incorporated into the amide nitrogen of L-glutamine (L-Gln) and then into L-glutamate (L-Glu). These results extend our previous observations in pine and potato that PAL-generated NH4+ is assimilated by the glutamine synthetase (GS; EC 6.3.1.2)/glutamate synthase (GOGAT; EC 1.4.1.13) pathway, with the NH4+ so formed ultimately being recycled back to L-Phe via L-Glu as aminoreceptor and donor.

  9. High Resolution 15N NMR of the 225 K Phase Transition of Ammonia Borane (NH3BH3): Mixed Order-Disorder and Displacive Behavior

    SciTech Connect

    Gunaydin-Sen, Ozge; Achey, Randall; Dalal, Nar S.; Stowe, Ashley C.; Autrey, Thomas

    2007-02-01

    We report high resolution 15N NMR probing of the solid-solid phase transition of 15N-labeled ammonia borane (NH3BH3) around 225 K. Both the 15N isotropic chemical shift, δiso, and the spin-lattice relaxation rate (T1-1) exhibited strong anomalies around 225 K. The analysis of T1-1 using the Bloembergen-Purcell and Pound model showed that the motional correlation time, τ, increased from about 1 ps to 100 ps while the corresponding Arrhenius activation energy increased from 6 to 13.4 kJ/mol on going through the transition. The observed strong temperature dependence of δiso was interpreted by an extension of the Bayer model. The time scale of the underlying motion was found to be in a reasonable agreement with the T1-1 data. These results imply that the NH3 rotor motion plays a pivotal role in the transition mechanism, and that the transition is of both order-disorder and displacive type. This work was supported by the Office of Basic Energy Sciences of the U. S. Department of Energy Chemical Sciences program. Pacific Northwest National Laboratory is operated by Battelle for the U. S. Department of Energy.

  10. Inoculation of Bacillus sphaericus UPMB-10 to Young Oil Palm and Measurement of Its Uptake of Fixed Nitrogen Using the 15N Isotope Dilution Technique

    PubMed Central

    Zakry, Fitri Abdul Aziz; Shamsuddin, Zulkifli H.; Rahim, Khairuddin Abdul; Zakaria, Zin Zawawi; Rahim, Anuar Abdul

    2012-01-01

    There are increasing applications of diazotrophic rhizobacteria in the sustainable agriculture system. A field experiment on young immature oil palm was conducted to quantify the uptake of N derived from N2 fixation by the diazotroph Bacillus sphaericus strain UPMB-10, using the 15N isotope dilution method. Eight months after 15N application, young immature oil palms that received 67% of standard N fertilizer application together with B. sphaericus inoculation had significantly lower 15N enrichment than uninoculated palms that received similar N fertilizers. The dilution of labeled N served as a marker for the occurrence of biological N2 fixation. The proportion of N uptake that was derived from the atmosphere was estimated as 63% on the whole plant basis. The inoculation process increased the N and dry matter yields of the palm leaflets and rachis significantly. Field planting of young, immature oil palm in soil inoculated with B. sphaericus UPMB-10 might mitigate inorganic fertilizer-N application through supplementation by biological nitrogen fixation. This could be a new and important source of nitrogen biofertilizer in the early phase of oil palm cultivation in the field. PMID:22446306

  11. 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

  12. Neutron capture cross section of {sup 15}N at stellar energies

    SciTech Connect

    Meissner, J.; Schatz, H.; Herndl, H.; Wiescher, M.; Beer, H.; Kaeppeler, F.

    1996-02-01

    The neutron capture rate on {sup 15}N may be of considerable importance for {ital s}-process nucleosynthesis in red giants as well as for the nucleosynthesis in inhomogeneous big bang scenarios. We measured the reaction cross section of {sup 15}N({ital n},{gamma}){sup 16}N at the Forschungszentrum Karlsruhe with a fast cyclic neutron activation technique at laboratory neutron energies of 25, 152, and 370 keV. Direct capture and shell model calculations were performed to interpret the results. The presented reaction rate is 30{endash}50{percent} smaller than the previously used theoretical rates. {copyright} {ital 1996 The American Physical Society.}

  13. 15N Fractionation in Star-Forming Regions and Solar System Objects

    NASA Technical Reports Server (NTRS)

    Wirstrom, Eva; Milam, Stefanie; Adande, GIlles; Charnley, Steven; Cordiner, Martin

    2015-01-01

    A central issue for understanding the formation and evolution of matter in the early Solar System is the relationship between the chemical composition of star-forming interstellar clouds and that of primitive Solar System materials. The pristinemolecular content of comets, interplanetary dust particles and carbonaceous chondrites show significant bulk nitrogen isotopic fractionation relative to the solar value, 14N15N 440. In addition, high spatial resolution measurements in primitive materials locally show even more extreme enhancements of 14N15N 100.

  14. A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides

    PubMed Central

    Acedo, Montse; Fàbrega, Carme; Aviño, Anna; Goodman, Myron; Fagan, Patricia; Wemmer, David; Eritja, Ramon

    1994-01-01

    The use of the ammonia deprotection step to introduce 15N labels at specific exocyclic amino positions of adenine, cytosine, guanine or 2-aminopurine of oligodeoxynucleotides is described. PMID:8065910

  15. Metabolism and excretion kinetics of {sup 14}C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites

    SciTech Connect

    Sukul, Premasis; Lamshoeft, Marc; Kusari, Souvik; Zuehlke, Sebastian; Spiteller, Michael

    2009-04-15

    Fluoroquinolones are amongst the most important antibiotics used in veterinary medicine. On this account the behavior of difloxacin (DIF) and its metabolites was investigated by administering the {sup 14}C-labeled and non-labeled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Sarafloxacin (SAR) was found to be the major metabolite, three further trace metabolites were also recovered, applying high-resolution (HR) mass spectrometric technique. The identification of DIF and SAR was confirmed by comparison with the spectroscopic and chromatographic data of the authentic references. The identification of the three trace metabolites was performed by HR-MS/MS. Only 8.1% of the administered radioactivity remained in the pig after 10 days and DIF accounted for 95.9% of the radioactivity excreted. More than 99% of the labeled compounds were detected and identified in the manure. The mean recoveries for all single electrolytes were {>=}94%. Linearity was established over concentration range 10-10,000 {mu}g/kg manure with a correlation coefficient {>=}0.99. By using in vitro antimicrobial activity tests against a group of standard pathogenic control strains, the results showed that the residual antibiotic concentrations in the manure of pigs are high enough to exhibit antibacterial activity.

  16. Metabolism and excretion kinetics of 14C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites.

    PubMed

    Sukul, Premasis; Lamshöft, Marc; Kusari, Souvik; Zühlke, Sebastian; Spiteller, Michael

    2009-04-01

    Fluoroquinolones are amongst the most important antibiotics used in veterinary medicine. On this account the behavior of difloxacin (DIF) and its metabolites was investigated by administering the (14)C-labeled and non-labeled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Sarafloxacin (SAR) was found to be the major metabolite, three further trace metabolites were also recovered, applying high-resolution (HR) mass spectrometric technique. The identification of DIF and SAR was confirmed by comparison with the spectroscopic and chromatographic data of the authentic references. The identification of the three trace metabolites was performed by HR-MS/MS. Only 8.1% of the administered radioactivity remained in the pig after 10 days and DIF accounted for 95.9% of the radioactivity excreted. More than 99% of the labeled compounds were detected and identified in the manure. The mean recoveries for all single electrolytes were 94%. Linearity was established over concentration range 10-10,000 microg/kg manure with a correlation coefficient 0.99. By using in vitro antimicrobial activity tests against a group of standard pathogenic control strains, the results showed that the residual antibiotic concentrations in the manure of pigs are high enough to exhibit antibacterial activity. PMID:19181312

  17. 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

  18. Whole-body protein turnover in preterm appropriate for gestational age and small for gestational age infants: comparison of [15N]glycine and [1-(13)C]leucine administered simultaneously.

    PubMed

    Van Goudoever, J B; Sulkers, E J; Halliday, D; Degenhart, H J; Carnielli, V P; Wattimena, J L; Sauer, P J

    1995-04-01

    Measurements of whole-body protein turnover in preterm infants have been made using different stable isotope methods. Large variation in results has been found, which could be due to different clinical conditions and/or the use of different tracers. We studied 14 appropriate for gestational age and nine small for gestational age orally fed preterm infants using [15N]glycine and [1-(13)C]leucine simultaneously, which allowed us to make a comparison of commonly used methods to calculate whole-body protein turnover. Whole-body protein turnover was calculated from 15N enrichment in urinary ammonia and urea after [15N]-glycine administration and from the 13C enrichment in expired CO2 after administration of [1-(13)C]leucine. Enrichment of alpha-ketoisocaproic acid after [1-(13)C]leucine constant infusion was measured as a direct parameter of whole-body protein turnover. Group means for whole-body protein turnover using [15N]glycine or [1-(13)C]leucine ranged from 10 to 14 g.kg-1.d-1, except when using the end product method that assumes a correlation between leucine oxidation and total nitrogen excretion. We found very low 15N enrichment of urinary urea in the majority of small for gestational age infants. These infants also had a lower nitrogen excretion in urine and oxidized less leucine. Nitrogen balance was higher in small for gestational age infants (416 +/- 25 mg.kg-1.d-1) compared with appropriate for gestational age infants (374 +/- 41 mg.kg-1.d-1, p = 0.003). [15N]Glycine does not seem to exchange its label with the body nitrogen pool to a significant degree and is therefore not always suitable as a carrier for 15N in protein turnover studies in premature infants. PMID:7596675

  19. Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by sup 15 N NMR spectroscopy

    SciTech Connect

    van Dijk, A.A.; de Lange, L.C.M.; Robillard, G.T. ); Bachovchin, W.W. )

    1990-09-04

    The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with {sup 15}N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with {sup 15}N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the N{delta}1 and N{epsilon}2 positions or at only the N{delta}1 position. {sup 15}N NMR spectra of two synthesized model compound, phosphoimidazole and phosphomethylimidazole, were also recorded. The authors show that, prior to phosphorylation, the protonated His15 N{epsilon}2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated N{delta}1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 N{delta}1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed.

  20. Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by 15N NMR spectroscopy.

    PubMed

    van Dijk, A A; de Lange, L C; Bachovchin, W W; Robillard, G T

    1990-09-01

    The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with 15N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with 15N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the N delta 1 and N epsilon 2 positions or at only the N delta 1 position. 15N NMR spectra of two synthesized model compounds, phosphoimidazole and phosphomethylimidazole, were also recorded. We show that, prior to phosphorylation, the protonated His15 N epsilon 2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated N delta 1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 N delta 1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed. PMID:2261470

  1. Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [15N]H4+ Assimilation in Lemna minor L

    PubMed Central

    Rhodes, David; Myers, Ann C.; Jamieson, Gene

    1981-01-01

    Rapid, sensitive, and selective methods for the determination of the 15N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [15N]H4+ assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate 15N abundance with an accuracy of ±1 atom% excess 15N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of 15N-labeled precursors. PMID:16662074

  2. Comparative effect of salinity on growth, grain yield, water use efficiency, δ(13)C and δ(15)N of landraces and improved durum wheat varieties.

    PubMed

    Chamekh, Zoubeir; Ayadi, Sawsen; Karmous, Chahine; Trifa, Youssef; Amara, Hajer; Boudabbous, Khaoula; Yousfi, Salima; Serret, Maria Dolors; Araus, José Luis

    2016-10-01

    Supplemental irrigation with low-quality water will be paramount in Mediterranean agriculture in the future, where durum wheat is a major crop. Breeding for salinity tolerance may contribute towards improving resilience to irrigation with brackish water. However, identification of appropriate phenotyping traits remains a bottleneck in breeding. A set of 25 genotypes, including 19 landraces and 6 improved varieties most cultivated in Tunisia, were grown in the field and irrigated with brackish water (6, 13 and 18dSm(-1)). Improved genotypes exhibited higher grain yield (GY) and water use efficiency at the crop level (WUEyield or 'water productivity'), shorter days to flowering (DTF), lower N concentration (N) and carbon isotope composition (δ(13)C) in mature kernels and lower nitrogen isotope composition (δ(15)N) in the flag leaf compared with landraces. GY was negatively correlated with DTF and the δ(13)C and N of mature kernels and was positively correlated with the δ(15)N of the flag leaf. Moreover, δ(13)C of mature kernels was negatively correlated with WUEyield. The results highlight the importance of shorter phenology together with photosynthetic resilience to salt-induced water stress (lower δ(13)C) and nitrogen metabolism (higher N and δ(15)N) for assessing genotypic performance to salinity. PMID:27593462

  3. Nitrate Removal in Two Relict Oxbow Urban Wetlands: A 15N Mass-balance Approach

    EPA Science Inventory

    A 15N-tracer method was used to quantify nitrogen (N) removal processes in two relict oxbow wetlands located adjacent to the Minebank Run restored stream reach in Baltimore County (Maryland, USA) during summer 2009 and early spring 2010. A mass-balance approach was used to determ...

  4. Using a Macroalgal δ15N Bioassay to Detect Cruise Ship Waste Water Effluent Inputs

    EPA Science Inventory

    Nitrogen stable isotopes are a powerful tool for tracking sources of N to marine ecosystems. I used green macroalgae as a bioassay organism to evaluate if the δ15N signature of cruise ship waste water effluent (CSWWE) could be detected in Skagway Harbor, AK. Opportunistic green...

  5. {sup 15}N(p,{alpha}{sub 0}){sup 12}C S factor

    SciTech Connect

    Barker, F. C.

    2008-10-15

    Experimental values of the astrophysical S factor for the {sup 15}N(p,{alpha}{sub 0}){sup 12}C reaction are available both from direct measurements and from the Trojan horse method. We here use R-matrix formulas to fit these values and to extrapolate to zero energy to obtain values of S(0)

  6. The use of delta(15)N in assessing sewage stress on coral reefs.

    PubMed

    Risk, Michael J; Lapointe, Brian E; Sherwood, Owen A; Bedford, Bradley J

    2009-06-01

    While coral reefs decline, scientists argue, and effective strategies to manage land-based pollution lag behind the extent of the problem. There is need for objective, cost-effective, assessment methods. The measurement of stable nitrogen isotope ratios, delta(15)N, in tissues of reef organisms shows promise as an indicator of sewage stress. The choice of target organism will depend upon study purpose, availability, and other considerations such as conservation. Algae are usually plentiful and have been shown faithfully to track sewage input. The organic matrix of bivalve shells can provide time series spanning, perhaps, decades. Gorgonians have been shown to track sewage, and can provide records potentially centuries-long. In areas where baseline data are lacking, which is almost everywhere, delta(15)N in gorgonians can provide information on status and trends. In coral tissue, delta(15)N combined with insoluble residue determination can provide information on both sewage and sediment stress in areas lacking baseline data. In the developed world, delta(15)N provides objective assessment in a field complicated by conflicting opinions. Sample handling and processing are simple and analysis costs are low. This is a method deserving widespread application. PMID:19286230

  7. 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.

  8. Tracing Nitrogen through Landscapes to Coastal Wetlands using d15N of Larval Fish

    EPA Science Inventory

    Our objective was to evaluate the use of the nitrogen stable isotope value (d15N) of larval fish as an indicator of incipient anthropogenic nitrogen loading to coastal wetlands in the Great Lakes. We sampled coastal wetlands in five Lake Superior south shore tributaries that had ...

  9. Monitoring the refinement of crystal structures with (15)N solid-state NMR shift tensor data.

    PubMed

    Kalakewich, Keyton; Iuliucci, Robbie; Mueller, Karl T; Eloranta, Harriet; Harper, James K

    2015-11-21

    The (15)N chemical shift tensor is shown to be extremely sensitive to lattice structure and a powerful metric for monitoring density functional theory refinements of crystal structures. These refinements include lattice effects and are applied here to five crystal structures. All structures improve based on a better agreement between experimental and calculated (15)N tensors, with an average improvement of 47.0 ppm. Structural improvement is further indicated by a decrease in forces on the atoms by 2-3 orders of magnitude and a greater similarity in atom positions to neutron diffraction structures. These refinements change bond lengths by more than the diffraction errors including adjustments to X-Y and X-H bonds (X, Y = C, N, and O) of 0.028 ± 0.002 Å and 0.144 ± 0.036 Å, respectively. The acquisition of (15)N tensors at natural abundance is challenging and this limitation is overcome by improved (1)H decoupling in the FIREMAT method. This decoupling dramatically narrows linewidths, improves signal-to-noise by up to 317%, and significantly improves the accuracy of measured tensors. A total of 39 tensors are measured with shifts distributed over a range of more than 400 ppm. Overall, experimental (15)N tensors are at least 5 times more sensitive to crystal structure than (13)C tensors due to nitrogen's greater polarizability and larger range of chemical shifts. PMID:26590548

  10. 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

  11. Variable δ15N Diet-Tissue Discrimination Factors among Sharks: Implications for Trophic Position, Diet and Food Web Models

    PubMed Central

    Olin, Jill A.; Hussey, Nigel E.; Grgicak-Mannion, Alice; Fritts, Mark W.; Wintner, Sabine P.; Fisk, Aaron T.

    2013-01-01

    The application of stable isotopes to characterize the complexities of a species foraging behavior and trophic relationships is dependent on assumptions of δ15N diet-tissue discrimination factors (∆15N). As ∆15N values have been experimentally shown to vary amongst consumers, tissues and diet composition, resolving appropriate species-specific ∆15N values can be complex. Given the logistical and ethical challenges of controlled feeding experiments for determining ∆15N values for large and/or endangered species, our objective was to conduct an assessment of a range of reported ∆15N values that can hypothetically serve as surrogates for describing the predator-prey relationships of four shark species that feed on prey from different trophic levels (i.e., different mean δ15N dietary values). Overall, the most suitable species-specific ∆15N values decreased with increasing dietary-δ15N values based on stable isotope Bayesian ellipse overlap estimates of shark and the principal prey functional groups contributing to the diet determined from stomach content analyses. Thus, a single ∆15N value was not supported for this speciose group of marine predatory fishes. For example, the ∆15N value of 3.7‰ provided the highest percent overlap between prey and predator isotope ellipses for the bonnethead shark (mean diet δ15N = 9‰) whereas a ∆15N value < 2.3‰ provided the highest percent overlap between prey and predator isotope ellipses for the white shark (mean diet δ15N = 15‰). These data corroborate the previously reported inverse ∆15N-dietary δ15N relationship when both isotope ellipses of principal prey functional groups and the broader identified diet of each species were considered supporting the adoption of different ∆15N values that reflect the predators’ δ15N-dietary value. These findings are critical for refining the application of stable isotope modeling approaches as inferences regarding a species’ ecological role in their

  12. Variable δ(15)N diet-tissue discrimination factors among sharks: implications for trophic position, diet and food web models.

    PubMed

    Olin, Jill A; Hussey, Nigel E; Grgicak-Mannion, Alice; Fritts, Mark W; Wintner, Sabine P; Fisk, Aaron T

    2013-01-01

    The application of stable isotopes to characterize the complexities of a species foraging behavior and trophic relationships is dependent on assumptions of δ(15)N diet-tissue discrimination factors (∆(15)N). As ∆(15)N values have been experimentally shown to vary amongst consumers, tissues and diet composition, resolving appropriate species-specific ∆(15)N values can be complex. Given the logistical and ethical challenges of controlled feeding experiments for determining ∆(15)N values for large and/or endangered species, our objective was to conduct an assessment of a range of reported ∆(15)N values that can hypothetically serve as surrogates for describing the predator-prey relationships of four shark species that feed on prey from different trophic levels (i.e., different mean δ(15)N dietary values). Overall, the most suitable species-specific ∆(15)N values decreased with increasing dietary-δ(15)N values based on stable isotope Bayesian ellipse overlap estimates of shark and the principal prey functional groups contributing to the diet determined from stomach content analyses. Thus, a single ∆(15)N value was not supported for this speciose group of marine predatory fishes. For example, the ∆(15)N value of 3.7‰ provided the highest percent overlap between prey and predator isotope ellipses for the bonnethead shark (mean diet δ(15)N = 9‰) whereas a ∆(15)N value < 2.3‰ provided the highest percent overlap between prey and predator isotope ellipses for the white shark (mean diet δ(15)N = 15‰). These data corroborate the previously reported inverse ∆(15)N-dietary δ(15)N relationship when both isotope ellipses of principal prey functional groups and the broader identified diet of each species were considered supporting the adoption of different ∆(15)N values that reflect the predators' δ(15)N-dietary value. These findings are critical for refining the application of stable isotope modeling approaches as inferences regarding a

  13. 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.

  14. Evaluating δ(15)N-body size relationships across taxonomic levels using hierarchical models.

    PubMed

    Reum, Jonathan C P; Marshall, Kristin N

    2013-12-01

    Ecologists routinely set out to estimate the trophic position of individuals, populations, and species composing food webs, and nitrogen stable isotopes (δ(15)N) are a widely used proxy for trophic position. Although δ(15)N values are often sampled at the level of individuals, estimates and confidence intervals are frequently sought for aggregations of individuals. If individual δ(15)N values are correlated as an artifact of sampling design (e.g., clustering of samples in space or time) or due to intrinsic groupings (e.g., life history stages, social groups, taxonomy), such estimates may be biased and exhibit overly optimistic confidence intervals. However, these issues can be accommodated using hierarchical modeling methods. Here, we demonstrate how hierarchical models offer an additional quantitative tool for investigating δ(15)N variability and we explicitly evaluate how δ(15)N varies with body size at successively higher levels of taxonomic aggregation in a diverse fish assemblage. The models take advantage of all available data, better account for uncertainty in parameters estimates, may improve inferences on coefficients corresponding to groups with small to moderate sample sizes, and partition variation across model levels, which provides convenient summaries of the 'importance' of each level in terms of unexplained heterogeneity in the data. These methods can easily be applied to diet-based studies of trophic position. Although hierarchical models are well-understood and established tools, their benefits have yet to be fully reaped by stable isotope and food web ecologists. We suggest that hierarchical models can provide a robust framework for conceptualizing and statistically modeling trophic position at multiple levels of aggregation. PMID:23812110

  15. 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.

  16. Solution NMR Experiment for Measurement of (15)N-(1)H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes.

    PubMed

    Eletsky, Alexander; Pulavarti, Surya V S R K; Beaumont, Victor; Gollnick, Paul; Szyperski, Thomas

    2015-09-01

    NMR residual dipolar couplings (RDCs) are exquisite probes of protein structure and dynamics. A new solution NMR experiment named 2D SE2 J-TROSY is presented to measure N-H RDCs for proteins and supramolecular complexes in excess of 200 kDa. This enables validation and refinement of their X-ray crystal and solution NMR structures and the characterization of structural and dynamic changes occurring upon complex formation. Accurate N-H RDCs were measured at 750 MHz (1)H resonance frequency for 11-mer 93 kDa (2)H,(15)N-labeled Trp RNA-binding attenuator protein tumbling with a correlation time τc of 120 ns. This is about twice as long as that for the most slowly tumbling system, for which N-H RDCs could be measured, so far, and corresponds to molecular weights of ∼200 kDa at 25 °C. Furthermore, due to the robustness of SE2 J-TROSY with respect to residual (1)H density from exchangeable protons, increased sensitivity at (1)H resonance frequencies around 1 GHz promises to enable N-H RDC measurement for even larger systems. PMID:26293598

  17. 1H, 13C and 15N Backbone Assignment of the EC-1 Domain of Human E-Cadherin

    PubMed Central

    Prasasty, Vivitri D.; Krause, Mary E.; Tambunan, Usman S. F.; Anbanandam, Asokan; Laurence, Jennifer S.; Siahaan, Teruna J.

    2014-01-01

    The EC1 domain of E-cadherin has been shown to be important for cadherin-cadherin homophilic interactions. Cadherins are responsible for calcium-mediated cell-cell adhesion located at the adherens junction of the biological barriers (i.e., intestinal mucosa and the blood-brain barrier (BBB). Cadherin peptides can modulate cadherin interactions to improve drug delivery through the blood-brain barriers (BBB). However, the mechanism of modulating the E-cadherin interactions by cadherin peptides has not been fully elucidated. To provide a basis for subsequent examination of the structure and peptide-binding properties of the EC1 domain of human E-cadherin using solution NMR spectroscopy, the 1H, 13C and 15N backbone resonance of the uniformly labeled-EC1 were assigned and the secondary structure was determined based on the chemical shift values. These resonance assignments are essential for assessing protein-ligand interactions and are reported here. PMID:24510398

  18. 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. PMID:26968868

  19. Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC–MS

    PubMed Central

    Basu, Sankha S; Deutsch, Eric C; Schmaier, Alec A; Lynch, David R; Blair, Ian A

    2014-01-01

    Background Intracellular metabolites such as CoA thioesters are modulated in a number of clinical settings. Their accurate measurement from surrogate tissues such as platelets may provide additional information to current serum and urinary biomarkers. Methods Freshly isolated platelets from healthy volunteers were treated with rotenone, propionate or isotopically labeled metabolic tracers. Using a recently developed LC–MS-based methodology, absolute changes in short-chain acyl-CoA thioesters were monitored, as well as relative metabolic labeling using isotopomer distribution analysis. Results Consistent with in vitro experiments, isolated platelets treated with rotenone showed decreased intracellular succinyl-CoA and increased β-hydroxybutyryl-CoA, while propionate treatment resulted in increased propionyl-CoA. In addition, isotopomers of the CoAs were readily detected in platelets treated with the [13C]- or [13C15N]-labeled metabolic precursors. Conclusion Here, we show that human platelets can provide a powerful ex vivo challenge platform with potential clinical diagnostic and biomarker discovery applications. PMID:24320127

  20. Mass Spectrometry-based Workflow for Accurate Quantification of Escherichia coli Enzymes: How Proteomics Can Play a Key Role in Metabolic Engineering*

    PubMed Central

    Trauchessec, Mathieu; Jaquinod, Michel; Bonvalot, Aline; Brun, Virginie; Bruley, Christophe; Ropers, Delphine; de Jong, Hidde; Garin, Jérôme; Bestel-Corre, Gwenaëlle; Ferro, Myriam

    2014-01-01

    Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics, and proteomics may be required to model the metabolism of potential cell factories. Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study, we developed a quantification workflow to analyze enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labeled standards with selected reaction monitoring analysis. First, full-length 15N labeled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimized for reproducibility and multiplexing capabilities over a single 30-min liquid chromatography-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimized for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and catalytic rate. This provides information that can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering. PMID:24482123

  1. REDOR NMR of stable-isotope-labeled protein binding sites

    SciTech Connect

    Schaefer, J.

    1994-12-01

    Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.

  2. Regional patterns of 15N natural abundance in forest ecosystems along a large transect in eastern China

    NASA Astrophysics Data System (ADS)

    Sheng, Wenping; Yu, Guirui; Fang, Huajun; Liu, Yingchun; Wang, Qiufeng; Chen, Zhi; Zhang, Li

    2014-02-01

    The regional determining factors underlying inter- and intra-site variation of 15N natural abundance in foliage, O horizon and mineral soil were investigated in eastern China.15N natural abundance values for these forest ecosystems were in the middle of the range of values previously found for global forest ecosystems. In contrast to commonly reported global patterns, temperate forest ecosystems were significantly more15N-enriched than tropical forest ecosystems, and foliage δ15N was negatively correlated with increasing mean annual temperature and net soil N mineralisation in eastern China. Tight N cycling in forest ecosystems and the use of atmospheric N deposition by trees might underlie the δ15N distribution patterns in eastern China. The existence of mycorrhizal fungi and root distribution profiles in the soil may also influence the15N natural abundance patterns in forest ecosystems of eastern China.

  3. Nitrogen cycling in an extreme hyperarid environment inferred from δ15N analyses of plants, soils and herbivore diet

    NASA Astrophysics Data System (ADS)

    Díaz, Francisca P.; Frugone, Matías; Gutiérrez, Rodrigo A.; Latorre, Claudio

    2016-03-01

    Climate controls on the nitrogen cycle are suggested by the negative correlation between precipitation and δ15N values across different ecosystems. For arid ecosystems this is unclear, as water limitation among other factors can confound this relationship. We measured herbivore feces, foliar and soil δ15N and δ13C values and chemically characterized soils (pH and elemental composition) along an elevational/climatic gradient in the Atacama Desert, northern Chile. Although very positive δ15N values span the entire gradient, soil δ15N values show a positive correlation with aridity as expected. In contrast, foliar δ15N values and herbivore feces show a hump-shaped relationship with elevation, suggesting that plants are using a different N source, possibly of biotic origin. Thus at the extreme limits of plant life, biotic interactions may be just as important as abiotic processes, such as climate in explaining ecosystem δ15N values.

  4. Nitrogen cycling in an extreme hyperarid environment inferred from δ(15)N analyses of plants, soils and herbivore diet.

    PubMed

    Díaz, Francisca P; Frugone, Matías; Gutiérrez, Rodrigo A; Latorre, Claudio

    2016-01-01

    Climate controls on the nitrogen cycle are suggested by the negative correlation between precipitation and δ(15)N values across different ecosystems. For arid ecosystems this is unclear, as water limitation among other factors can confound this relationship. We measured herbivore feces, foliar and soil δ(15)N and δ(13)C values and chemically characterized soils (pH and elemental composition) along an elevational/climatic gradient in the Atacama Desert, northern Chile. Although very positive δ(15)N values span the entire gradient, soil δ(15)N values show a positive correlation with aridity as expected. In contrast, foliar δ(15)N values and herbivore feces show a hump-shaped relationship with elevation, suggesting that plants are using a different N source, possibly of biotic origin. Thus at the extreme limits of plant life, biotic interactions may be just as important as abiotic processes, such as climate in explaining ecosystem δ(15)N values. PMID:26956399

  5. Nitrogen cycling in an extreme hyperarid environment inferred from δ15N analyses of plants, soils and herbivore diet

    PubMed Central

    Díaz, Francisca P.; Frugone, Matías; Gutiérrez, Rodrigo A.; Latorre, Claudio

    2016-01-01

    Climate controls on the nitrogen cycle are suggested by the negative correlation between precipitation and δ15N values across different ecosystems. For arid ecosystems this is unclear, as water limitation among other factors can confound this relationship. We measured herbivore feces, foliar and soil δ15N and δ13C values and chemically characterized soils (pH and elemental composition) along an elevational/climatic gradient in the Atacama Desert, northern Chile. Although very positive δ15N values span the entire gradient, soil δ15N values show a positive correlation with aridity as expected. In contrast, foliar δ15N values and herbivore feces show a hump-shaped relationship with elevation, suggesting that plants are using a different N source, possibly of biotic origin. Thus at the extreme limits of plant life, biotic interactions may be just as important as abiotic processes, such as climate in explaining ecosystem δ15N values. PMID:26956399

  6. Isotopic variability of cave bears (δ15N, δ13C) across Europe during MIS 3

    NASA Astrophysics Data System (ADS)

    Krajcarz, Magdalena; Pacher, Martina; Krajcarz, Maciej T.; Laughlan, Lana; Rabeder, Gernot; Sabol, Martin; Wojtal, Piotr; Bocherens, Hervé

    2016-01-01

    Collagen, the organic fraction of bone, records the isotopic parameters of consumed food for carbon (δ13C) and nitrogen (δ15N). This relationship of isotopic signature between diet and tissue is an important tool for the study of dietary preferences of modern and fossil animal species. Since the first information on the isotopic signature of cave bear was reported, numerous data from Europe have become available. The goal of this work is to track the geographical variation of cave bear collagen isotopic values in Europe during Marine Isotopic Stage 3 (about 60,000-25,000 yr BP). In this study the results of new δ13C and δ15N isotopic analyses of cave bear collagen from four Central-Eastern European sites are presented, as well as a review of all published isotopic data for cave bears of the same period. The main conclusion is a lack of geographical East-West pattern in the variations of δ13C and δ15N values of cave bear collagen. Moreover, no relationship was found between cave bear taxonomy and isotopic composition. The cave bears from Central-Eastern Europe exhibit δ13C and δ15N values near the average of the range of Central, Western and Southern European cave bears. Despite the fact that most cave bear sites follow an altitudinal gradient, separate groups of sites exhibit shift in absolute values of δ13C, what disturbs an altitude-related isotopic pattern. The most distinct groups are: high Alpine sites situated over 1500 m a.s.l. - in terms of δ13C; and two Romanian sites Peştera cu Oase and Urşilor - in case of δ15N. Although the cave bear isotopic signature is driven by altitude, the altitudinal adjustment of isotopic data is not enough to explain the isotopic dissimilarity of these cave bears. The unusually high δ15N signature of mentioned Romanian sites is an isolated case in Europe. Cave bears from relatively closely situated Central-Eastern European sites and other Romanian sites are more similar to Western European than to Romanian

  7. Glycolate metabolism in low and high CO sub 2 -grown chlorella pyrenoidosa and Pavlova lutheri as determined by sup 18 O-labeling

    SciTech Connect

    de Veau, E.J.; Burris, J.E. )

    1989-11-01

    Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring {sup 18}O-labeled intermediates of the glycolate pathway. Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from {sup 18}O-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high CO{sub 2}-grown cells than in air-grown cells when both were assayed under the same O{sub 2} and CO{sub 2} concentrations. Synthesis of glycolate, for both types of cells, was stimulated by high O{sub 2} levels and inhibited by high CO{sub 2} levels. Glycolate synthesis in 1.5% CO{sub 2}-grown Chlorella, when exposed to a 0.035% CO{sub 2} atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the O{sub 2} concentration was increased from 21 to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the O{sub 2} concentration was lowered to 2% or the CO{sub 2}-concentration was raised to 1.5%. Glycolate excretion was also sensitive to O{sub 2} and CO{sub 2} concentrations in 1.5% CO{sub 2}-grown cells and the glycolate that was excreted was {sup 18}O-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella. Photorespiratory {sup 18}O-labeling kinetics were determined for Pavlova lutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.

  8. Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.

    PubMed

    Okar, D A; Live, D H; Devany, M H; Lange, A J

    2000-08-15

    The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were labeled with (15)N, both specifically at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated (15)N resonances were assigned by correlation to the C2' protons which had been assigned previously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the (1)H-(15)N HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation of the phosphate binding site filled by the O2 phosphate of the substrate, fructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermediate. The phosphohistidine intermediate is characterized by two hydrogen bonds involving the catalytic histidines, His-258 and His-392, which are directly observed at the N1' positions of the imidazole rings. The N1' of phospho-His-258 is protonated ((1)H chemical shift, 14.0 ppm) and hydrogen bonded to the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded ((1)H chemical shift, 13.5 ppm) to the phosphoryl moiety of the phosphohistidine. The existence of a protonated phospho-His-258 intermediate and the observation of a fairly strong hydrogen bond to the same phosphohistidine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as probes of the catalytic site mutation of Glu-327 to alanine revealed that, in addition to its function as the proton donor to fructose-6-phosphate during formation of the transient phosphohistidine intermediate at the N3' of His-258, this residue has a significant role in maintaining the structural integrity of the catalytic site. The (1)H-(15)N HSQC data also provide clear evidence that despite being a surface residue, His-446 has a very acidic pK(a), much less than 6.0. On the basis of

  9. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  10. Natural abundance 14N and 15N solid-state NMR of pharmaceuticals and their polymorphs

    DOE PAGESBeta

    Veinberg, Stanislav L.; Johnston, Karen E.; Jaroszewicz, Michael J.; Kispal, Brianna M.; Mireault, Christopher R.; Kobayashi, Takeshi; Pruski, Marek; Schurko, Robert W.

    2016-06-08

    14N ultra-wideline (UW), 1H{15N} indirectly-detected HETCOR (idHETCOR) and 15N dynamic nuclear polarization (DNP) solid-state NMR (SSNMR) experiments, in combination with plane-wave density functional theory (DFT) calculations of 14N EFG tensors, were utilized to characterize a series of nitrogen-containing active pharmaceutical ingredients (APIs), including HCl salts of scopolamine, alprenolol, isoprenaline, acebutolol, dibucaine, nicardipine, and ranitidine. Here, a case study applying these methods for the differentiation of polymorphs of bupivacaine HCl is also presented. All experiments were conducted upon samples with naturally-abundant nitrogen isotopes. For most of the APIs, it was possible to acquire frequency-stepped UW 14N SSNMR spectra of stationarymore » samples, which display powder patterns corresponding to pseudo-tetrahedral (i.e., RR'R"NH+ and RR'NH2+) or other (i.e., RNH2 and RNO2) nitrogen environments.« less

  11. Interference effects in proton scattering on {sup 15}N nuclei at intermediate energies

    SciTech Connect

    Ibraeva, E. T.; Zhusupov, M.A.; Imambekov, O.; Krassovitskiy, P. M.

    2010-08-15

    The differential cross section for proton scattering on {sup 15}N nuclei is calculated within Glauber diffraction theory at energies of 0.2, 0.6, and 1.0 GeV. Use is made of the shell-model wave function for the {sup 15}N nucleus. The contribution of single and double collisions to the Glauber operator {Omega} is taken into account. The sensitivity of the differential cross sections to the contributions of scattering on nucleons from different shells, to the parameters of the elementary pN amplitude, and to the energy of projectile protons is investigated. It is shown that the interference between amplitudes corresponding to different collision multiplicities, as well as between the amplitudes for scattering on nucleons from different shells, determines special features of the cross section.

  12. 1H, 13C and 15N NMR assignments of phenazopyridine derivatives.

    PubMed

    Burgueño-Tapia, Eleuterio; Mora-Pérez, Yolanda; Morales-Ríos, Martha S; Joseph-Nathan, Pedro

    2005-03-01

    Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were studied by one- and two-dimensional (1)H, (13)C and (15)N NMR methodology. The assignments, combined with DFT calculations, reveal that the preferred protonation site of the drug is the pyridine ring nitrogen atom. The chemoselective acetylation of phenazopyridine (2) and its influence on the polarization of the azo nitrogen atoms were evidenced by the (15)N NMR spectra. Molecular calculations of the phenazopyridines 2-4 show that the pyridine and phenyl groups are oriented in an antiperiplanar conformation with intramolecular hydrogen bonding between the N-b atom and the C-2 amino group preserving the E-azo stereochemistry. PMID:15625718

  13. Catalytic Roles of βLys87 in Tryptophan Synthase: 15N Solid State NMR Studies

    PubMed Central

    Caulkins, Bethany G.; Yang, Chen; Hilario, Eduardo; Fan, Li; Dunn, Michael F.; Mueller, Leonard J.

    2015-01-01

    The proposed mechanism for tryptophan synthase shows βLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4′ for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid-base catalysis). ε-15N-lysine TS was prepared to access the protonation state of βLys87 using 15N solid-state nuclear magnetic resonance (SSNMR) spectroscopy for three quasi-stable intermediates along the reaction pathway. These experiments establish that the protonation state of the ε-amino group switches between protonated and neutral states as the β-site undergoes conversion from one intermediate to the next during catalysis, corresponding to mechanistic steps where this lysine residue has been anticipated to play alternating acid and base catalytic roles that help steer reaction specificity in tryptophan synthase catalysis. PMID:25688830

  14. Mycorrhizal Fungi Provide Most of the Nitrogen for Symbiotic Arctic Plants: 15N Evidence

    NASA Astrophysics Data System (ADS)

    Hobbie, J. E.; Hobbie, E. A.

    2004-12-01

    When soil nitrogen is in short supply, most terrestrial plants form symbioses with fungi (mycorrhizae) in which fine hyphal threads take up soil nitrogen, transport it into plant roots, and in return receive plant sugars. Because the transfer rates are very difficult to measure in nature, ecologists need new tools by which to assess the role of mycorrhizal fungi in carbon and nitrogen cycling. Recent studies indicate that the natural abundance of 15N taken up from the soil by hyphae is changed during transfer of nitrogen to roots; the result is large differences among the natural abundance of 15N in soil, symbiotic plants, and symbiotic fungi that depend on the mass balance of nitrogen in the mycorrhizal symbiosis. Measurements were carried out in acidic tussock tundra at the Toolik Lake LTER site in Arctic Alaska (68\\deg N 149\\deg W). The \\delta15N of soil N was 1.5%, of soil ammonium was 1.5%, of ericoid and ectomycorrhizal plants was -5.0%, and of ectomycorrhizal fungi was 7.0 parts per mille%. The mass balance of the 15N shows that the plants received 61-86% of their nitrogen from the fungal hyphae. These values, when combined with known plant growth rates, reveal that the plants provided 7-16% of their photosynthetic carbon to the fungi for growth and respiration, or about 25% of all carbon allocated to belowground processes. This analytical technique could be readily applied to other nitrogen-limited ecosystems such as many temperate and boreal forests to quantify the importance for terrestrial carbon and nitrogen cycling of mycorrhizally mediated transfers at the plant-soil interface.

  15. δ15N in the turtle grass from the Mexican Caribbean

    NASA Astrophysics Data System (ADS)

    Talavera-Saenz, A.; Sanchez, A.; Ortiz-Hernandez, M.

    2013-05-01

    Nutrient inputs associated with population growth threaten the integrity of coastal ecosystems. To assess the rapid increase in tourism, we compared the δ15N from Thalassia testudinum collected at sites with different levels of tourism development and population to detect the N inputs of wastewater discharge (WD) along the coast of Quintana Roo. The contributions of nitrogen enriched in 15N are directly related to the increase of WD inputs in areas of high tourism development (Nichupte Lagoon in Cancun, >3 million tourists per year from 2007 to 2011 and 0.7 million of resident population) and decreased towards Bahia Akumal and Tulum (>3 million tourists per year from 2007 to 2011 and 0.15 million of resident population). The δ15N from T. testudinum was significantly lower at Mahahual and Puerto Morelos (about 0.4 million tourists per year in 2007 to 2011 and 0.25 million of resident population) than other the sites. In areas of the lowest development and with tourist activity restricted and small population, such as the Yum Balam Reserve and Sian Ka'an Biosphere Reserve, the δ15N values were in much higher enrichment that Mahahual and Puerto Morelos. Therefore is suggested that Mahahual and Puerto Morelos may be used for baseline isotopic monitoring, over environmental pressure on the reef lagoon ecosystem, where tourist activities and population are growing very slow rate. The anthropogenic N input has the potential to impact, both environmentally and economically, the seagrass meadows and the coral reefs along the coast of Quintana Roo and the Caribbean.

  16. δ15N as a Potential Paleoenvironmental Proxy for Nitrogen Loading in Chesapeake Bay

    NASA Astrophysics Data System (ADS)

    Black, H. D.; Andrus, C. F.; Rick, T.; Hines, A.

    2013-12-01

    Stable isotope analysis of Eastern Oyster (Crassostrea virginica) and other mollusk shells from archaeological sites is a useful means of acquiring paleoenvironmental data. Recently, nitrogen isotopes have been identified as a potential new proxy in these shells. δ15N content in mollusk shells is affected by numerous anthropogenic and natural influences and may be used as an environmental proxy for nitrogen loading conditions. Chesapeake Bay is well known for both historic and modern pollution problems from numerous anthropogenic sources, such as fertilizer runoff, sewage discharge, and densely populated land use and serves as an ideal study location for long-term nitrogen loading processes. Longer records of these processes may be recorded in abundant archaeological remains around the bay, however, little is known about the stability of δ15N and %N in shell material over recent geologic time. In this study, 90 archaeological C. virginica shells were collected by the Smithsonian Institution from the Rhode River Estuary within Chesapeake Bay and range in age from ~150 to 3200 years old. Twenty-two modern C. virginica shells were also collected from nearby beds in the bay. All shell samples were subsampled from the resilifer region of the calcitic shell using a hand-held micro drill and were analyzed using EA-IRMS analysis to determine the potential temporal variability of δ15N and %N as well as creating a baseline for ancient nitrogen conditions in the bay area. Modern POM water samples and C. virginica soft tissues were also analyzed in this study to determine the degree of seasonal variation of δ15N and %N in Chesapeake Bay.

  17. SEARCH FOR AN 80-ms SPONTANEOUS FISSION ACTIVITY IN BOMBARDMENTS OF 249Bk WITH 15N

    SciTech Connect

    Nitschke, J.M.; Fowler, M.; Ghiorso, A.; Leber, R.E.; Nurmia, M.J.; Somerville, L.P.; Williams, K.E.; Hulet, E.K.; Landrum, J.H.; Lougheed, R.W.; Wild, J.F.; Bemis, Jr., C.E.; Silva, R.J.; Eskola, P.

    1980-01-01

    A rotating drum system was used to search for an 80-ms spontaneous fission (sf) activity in the reaction of {sup 15}N with {sup 249}Bk. No such activity was found beyond a cross section limit of 0.3 {+-} 0.3 nb. A sf activity with a half-life of about 20 ms and a formation cross section of 12 nb at 82 MeV was observed. The identity of this activity has not been determined.

  18. Determination of the δ15N of total nitrogen in solids; RSIL lab code 2893

    USGS Publications Warehouse

    Revesz, Kinga; Qi, Haiping; Coplen, Tyler B.

    2006-01-01

    The purpose of the Reston Stable Isotope Laboratory (RSIL) lab code 2893 is to determine the δ(15N/14N), abbreviated as δ15N , of total nitrogen in solid samples. A Carlo Erba NC 2500 elemental analyzer (EA) is used to convert total nitrogen in a solid sample into N2 gas. The EA is connected to a continuous flow isotope-ratio mass spectrometer (CF-IRMS), which determines relative difference in the isotope-amount ratios of stable nitrogen isotopes (15N/14N)of the product N2 gas. The combustion is quantitative; no isotopic fractionation is involved. Samples are placed in a tin capsule and loaded into the Costech Zero Blank Autosampler of the EA. Under computer control, samples are dropped into a heated reaction tube that contains an oxidant, where the combustion takes place in a helium atmosphere containing an excess of oxygen gas. Combustion products are transported by a helium carrier through a reduction tube to remove excess oxygen and convert all nitrous oxides into N2 and through a drying tube to remove water. The gas-phase products, mainly CO2 and N2, are separated by a gas chromatograph. The gas is then introduced into the isotope-ratio mass spectrometer (IRMS) through a Finnigan MAT (now Thermo Scientific) ConFlo II interface, which also is used to inject N2 reference gas and helium for sample dilution. The IRMS is a Thermo Scientific Delta V Plus CF-IRMS. It has a universal triple collector, two wide cups with a narrow cup in the middle, capable of measuring mass/charge (m/z) 28, 29, 30, simultaneously. The ion beams from N2 are as follows: m/z 28 = N2 = 14N14N; m/z 29 = N2 = 14N15N primarily; m/z 30 = NO = 14N16O primarily, which is a sign of contamination or incomplete reduction.

  19. Community 15N isoscapes to resolve plant-plant-interactions at the spatial scale

    NASA Astrophysics Data System (ADS)

    Hellmann, Christine; Rascher, Katherine G.; Máguas, Cristina; Werner, Christiane

    2014-05-01

    Isoscapes have greatly improved our ability to understand biogeochemical processes on continental to global scales. However, the isoscapes framework may also have significant potential to resolve the spatial component of within-community interactions. For example, exotic plant invaders often exert strong impacts on ecosystem functioning, particularly regarding water-, carbon- and nutrient-cycles, but the spatial extent of such alterations is largely unknown. Here we show that massive N input by the N2-fixing exotic invasive Acacia longifolia to a Portuguese dune system can be traced using spatially resolved information on native plants' leaf δ15N. We found isotopic signatures of N to differ strongly between the native system (δ15N c. -10 o) and the atmospherically derived N in A. longifolia phyllodes (δ15N c. 0 o). Thus, sources of N for native plants could be readily distinguished. Leaf δ15N of a native, non-fixing species was increasingly enriched the closer the plant grew to the invader, indicating uptake of fixed N provided by A. longifolia. The enrichment was evident far beyond the stands of the invader, demonstrating that A. longifolia affected N budgets of native species up to a distance of 8 m exceeding the margin of the canopy. Furthermore, using the isoscapes approach, we were able to quantify the total area of N enrichment and could thus show that the area affected by invasion was at least 3.5 times larger than the area actually occupied by the invader. However, a native N2-fixing species had no such effects. Thus, downscaling isoscapes to the community level opens new frontiers in quantifying the spatial dimension of functional changes associated with plant invasions. Moreover, considering the feasibility and applicability of this approach, it may provide a promising tool to identify, quantify and monitor different types of functional plant-plant interactions within communities at a spatially explicit scale.

  20. Food webs of two intermittently open estuaries receiving 15N-enriched sewage effluent

    NASA Astrophysics Data System (ADS)

    Hadwen, Wade L.; Arthington, Angela H.

    2007-01-01

    Carbon and nitrogen stable isotope signatures were used to assess the response of food webs to sewage effluent discharged into two small intermittently open estuaries in northern New South Wales, Australia. One of these systems, Tallows Creek, has a history of direct sewage inputs, whilst the other, Belongil Creek, receives wastewater via an