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Sample records for 13c label incorporation

  1. Application of 13C isotope labeling using liquid chromatography mass spectrometry (LC-MS) to determining phosphate-containing metabolic incorporation

    PubMed Central

    Bhowmik, Salil Kumar; Putluri, Vasanta; Kommagani, Ramakrishna; Konde, Sai Aparna; Lydon, John P.; Sreekumar, Arun; Putluri, Nagireddy

    2016-01-01

    Here, we describe an approach wherein negative electrospray ionization mass spectrometry has used to understand the relative flux through phosphate containing metabolic intermediates associated with central carbon metabolism after administering cells with 13C-labeled substrates. The method was applied to examine the 13C incorporation through glycolysis in T47D breast cancer cells and showed reduction of glycolytic relative flux upon treatment with 2-Deoxyglucose. PMID:24338880

  2. Lack of 13C-label incorporation suggests low turnover rates of thaumarchaeal intact polar tetraether lipids in sediments from the Iceland Shelf

    NASA Astrophysics Data System (ADS)

    Lengger, S. K.; Lipsewers, Y. A.; de Haas, H.; Sinninghe Damsté, J. S.; Schouten, S.

    2013-08-01

    Thaumarchaeota are amongst the most abundant microorganisms in aquatic environments, however, their metabolism in marine sediments is still debated. Labeling studies in marine sediments have previously been undertaken, but focused on complex organic carbon substrates which Thaumarchaeota have not yet been shown to take up. In this study, we investigated the activity of Thaumarchaeota in sediments by supplying different 13C-labeled substrates which have previously been shown to be incorporated into archaeal cells in water incubations and/or enrichment cultures. We determined the incorporation of 13C-label from bicarbonate, pyruvate, glucose and amino acids into thaumarchaeal intact polar lipid-glycerol dibiphytanyl glycerol tetraethers (IPL-GDGTs) during 4-6 day incubations of marine sediment cores from three different sites on the Iceland Shelf. Thaumarchaeal intact polar lipids were detected at all stations and concentrations remained constant or decreased slightly upon incubation. No 13C incorporation in any IPL-GDGT was observed at stations 2 (clay-rich sediment) and 3 (organic-rich sediment). In bacterial/eukaryotic IPL-derived fatty acids at station 3, contrastingly, a large uptake of 13C label (up to +80‰) was found. 13C was also respired during the experiment as shown by a substantial increase in the 13C content of the dissolved inorganic carbon. In IPL-GDGTs recovered from the sandy sediments at station 1, however, some enrichment in 13C (1-4‰) was detected after incubation with bicarbonate and pyruvate. The low incorporation rates suggest a low activity of Thaumarchaeota in marine sediments and/or a low turnover rate of thaumarchaeal IPL-GDGTs due to their low degradation rates. Cell numbers and activity of sedimentary Thaumarchaeota based on IPL-GDGT measurements may thus have previously been overestimated.

  3. Lack of 13C-label incorporation suggests low turnover rates of thaumarchaeal intact polar tetraether lipids in sediments from the Iceland shelf

    NASA Astrophysics Data System (ADS)

    Lengger, S. K.; Lipsewers, Y. A.; de Haas, H.; Sinninghe Damsté, J. S.; Schouten, S.

    2014-01-01

    Thaumarchaeota are amongst the most abundant microorganisms in aquatic environments, however, their metabolism in marine sediments is still debated. Labeling studies in marine sediments have previously been undertaken, but focused on complex organic carbon substrates which Thaumarchaeota have not yet been shown to take up. In this study, we investigated the activity of Thaumarchaeota in sediments by supplying different 13C-labeled substrates which have previously been shown to be incorporated into archaeal cells in water incubations and/or enrichment cultures. We determined the incorporation of 13C-label from bicarbonate, pyruvate, glucose and amino acids into thaumarchaeal intact polar lipid-glycerol dibiphytanyl glycerol tetraethers (IPL-GDGTs) during 4-6 day incubations of marine sediment cores from three sites on the Iceland shelf. Thaumarchaeal intact polar lipids, in particular crenarchaeol, were detected at all stations and concentrations remained constant or decreased slightly upon incubation. No 13C incorporation in any IPL-GDGT was observed at stations 2 (clay-rich sediment) and 3 (organic-rich sediment). In bacterial/eukaryotic IPL-derived fatty acids at station 3, contrastingly, a large uptake of 13C label (up to + 80‰ ) was found. 13C was also respired during the experiment as shown by a substantial increase in the 13C content of the dissolved inorganic carbon. In IPL-GDGTs recovered from the sandy sediments at station 1, however, some enrichment in δ13C (1-4‰ ) was detected after incubation with bicarbonate and pyruvate. The low incorporation rates suggest a low activity of Thaumarchaeota in marine sediments and/or a low turnover rate of thaumarchaeal IPL-GDGTs due to their low degradation rates. Cell numbers and activity of sedimentary Thaumarchaeota based on IPL-GDGT measurements may thus have previously been overestimated.

  4. Incorporation of {sup 13}C-labeled intermediates into developing lignin revealed by analytical pyrolysis and CuO oxidation in combination with IRM-GC-MS

    SciTech Connect

    Eglinton, T.I.; Goni, M.A.; Boon, J.J.

    1995-12-31

    Tissue samples from Ginkgo shoots (Ginkgo biloba L.) and Rice grass (Oryzasitiva sp.) incubated in the presence of {sup 13}C-labeled substrates such as coniferin (postulated to be biosynthetic intermediates in lignin biosynthesis) were studied using thermal and chemical dissociation methods in combination with molecular-level isotopic measurements. The aim of the study was (1) to investigate dissociation mechanisms, and (2) to examine and quantify the proportions of labeled material incorporated within each sample. Isotopic analysis of specific dissociation products revealed the presence of the label in its original positions, and only within lignin-derived (phenolic) products. Moreover, the distribution and isotopic composition of the dissociation products strongly suggest an origin from newly-formed lignin. These results clearly indicate that there is no {open_quotes}scrambling{close_quotes} of carbon atoms as a result of the dissociation process, thereby lending support to this analytical approach. In addition, the data provide confidence in the selective labeling approach for elucidation of the structure and biosynthesis of lignin.

  5. Incorporation of 13C labelled root-shoot residues in soil in the presence of Lumbricus terrestris: An isotopic and molecular approach

    NASA Astrophysics Data System (ADS)

    Vidal, Alix; Alexis, Marie; Nguyen Tu, Thanh Tu; Anquetil, Christelle; Vaury, Véronique; Derenne, Sylvie; Quenea, Katell

    2016-04-01

    Litter from plant biomass deposited on soil surface can either be mineralized; releasing CO2 to the atmosphere, or transferred into the soil as organic compounds. Both pathways depend on biotic factors such as litter characteristics and the of soil organism activity. During the last decades, many studies have focused on the origin of organic matter, with a particular attention to the fate of root and shoot litter. It is generally admitted that roots decompose at a slower rate than shoots, resulting in a higher carbon sequestration in soil for compounds originating from roots. Earthworms play a central role in litter decomposition and carbon cycling, ingesting both organic and mineral compounds which are mixed, complexed and dejected in the form of casts at the soil surface or along earthworm burrows. The simultaneous impact of earthworms and root-shoot on soil carbon cycling is still poorly understood. This study aimed at (1) defining the rate of incorporation of root and shoot litter with or without earthworms and (2) characterizing the molecular composition of soil organic matter upon litter decomposition, after one year of experimentation. A mesocosm experiment was set up to follow the incorporation of 13C labelled Ryegrass root and shoot litter in the soil, in the presence of anecic earthworms (Lumbricus terrestris). Soil samples were collected at 0-20 and 40-60 cm, as well as surface casts, at the beginning and after 1, 2, 4, 8, 24 and 54 weeks of experiment. Organic carbon content and δ13C values were determined for all the samples with Elemental Analysis - Isotope Ratio Mass Spectrometry. Lipid-free soil and cast samples after 54 weeks of incubation were analyzed with Pyrolysis-Gas Chromatography-Mass Spectrometry. Pyrolysis products were grouped into six classes: polysaccharides, lignin derived compounds, phenols, N-compounds, aliphatic compounds and sterols. Each pyrolysis product was quantified thanks to its peak area, relative to the total area of the

  6. 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

  7. NMR structure analysis of uniformly 13C-labeled carbohydrates.

    PubMed

    Fontana, Carolina; Kovacs, Helena; Widmalm, Göran

    2014-06-01

    In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of (13)C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly (13)C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-(13)C)-sucrose, 342 Da] and one compound of medium molecular weight ((13)C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The (13)C resonances are traced using (13)C-(13)C correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the (13)C resonances, the (1)H chemical shifts are derived in a straightforward manner using one-bond (1)H-(13)C correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J CC splitting of the (13)C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either (13)C or (1)H detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the (1)H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the (13)C-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with (15)N at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and (13)C-detected (H)CACO spectra.

  8. NMR structure analysis of uniformly 13C-labeled carbohydrates.

    PubMed

    Fontana, Carolina; Kovacs, Helena; Widmalm, Göran

    2014-06-01

    In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of (13)C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly (13)C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-(13)C)-sucrose, 342 Da] and one compound of medium molecular weight ((13)C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The (13)C resonances are traced using (13)C-(13)C correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the (13)C resonances, the (1)H chemical shifts are derived in a straightforward manner using one-bond (1)H-(13)C correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J CC splitting of the (13)C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either (13)C or (1)H detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the (1)H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the (13)C-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with (15)N at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and (13)C-detected (H)CACO spectra. PMID:24771296

  9. Experimental design principles for isotopically instationary 13C labeling experiments.

    PubMed

    Nöh, Katharina; Wiechert, Wolfgang

    2006-06-01

    13C metabolic flux analysis (MFA) is a well-established tool in Metabolic Engineering that found numerous applications in recent years. However, one strong limitation of the current method is the requirement of an-at least approximate-isotopic stationary state at sampling time. This requirement leads to a principle lower limit for the duration of a 13C labeling experiment. A new methodological development is based on repeated sampling during the instationary transient of the 13C labeling dynamics. The statistical and computational treatment of such instationary experiments is a completely new terrain. The computational effort is very high because large differential equations have to be solved and, moreover, the intracellular pool sizes play a significant role. For this reason, the present contribution works out principles and strategies for the experimental design of instationary experiments based on a simple example network. Hereby, the potential of isotopically instationary experiments is investigated in detail. Various statistical results on instationary flux identifiability are presented and possible pitfalls of experimental design are discussed. Finally, a framework for almost optimal experimental design of isotopically instationary experiments is proposed which provides a practical guideline for the analysis of large-scale networks.

  10. An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris.

    PubMed

    Rodriguez, E; Krishna, N R

    2001-07-01

    We report a new and cost-effective approach to prepare (15)N/(13)C labeled proteins for NMR using the Pichia pastoris expression system. Four protocols (P1 to P4) were defined and compared using recombinant Ovine interferon-tau (rOvIFN-tau). Our results demonstrate that in order to get full incorporation of (15)N and (13)C, the isotopes are not totally required during the initial growth phase of P. pastoris culture. The addition of small amounts of (15)N and (13)C compounds 6 h prior to the methanol induction phase is sufficient to obtain 99% incorporation of heavy isotopes into the protein. Our optimized protocol P4 is two-thirds less costly than the classical method using (15)N and (13)C isotopes during the entire growth phase.

  11. A Method to Constrain Genome-Scale Models with 13C Labeling Data

    PubMed Central

    García Martín, Héctor; Kumar, Vinay Satish; Weaver, Daniel; Ghosh, Amit; Chubukov, Victor; Mukhopadhyay, Aindrila; Arkin, Adam; Keasling, Jay D.

    2015-01-01

    Current limitations in quantitatively predicting biological behavior hinder our efforts to engineer biological systems to produce biofuels and other desired chemicals. Here, we present a new method for calculating metabolic fluxes, key targets in metabolic engineering, that incorporates data from 13C labeling experiments and genome-scale models. The data from 13C labeling experiments provide strong flux constraints that eliminate the need to assume an evolutionary optimization principle such as the growth rate optimization assumption used in Flux Balance Analysis (FBA). This effective constraining is achieved by making the simple but biologically relevant assumption that flux flows from core to peripheral metabolism and does not flow back. The new method is significantly more robust than FBA with respect to errors in genome-scale model reconstruction. Furthermore, it can provide a comprehensive picture of metabolite balancing and predictions for unmeasured extracellular fluxes as constrained by 13C labeling data. A comparison shows that the results of this new method are similar to those found through 13C Metabolic Flux Analysis (13C MFA) for central carbon metabolism but, additionally, it provides flux estimates for peripheral metabolism. The extra validation gained by matching 48 relative labeling measurements is used to identify where and why several existing COnstraint Based Reconstruction and Analysis (COBRA) flux prediction algorithms fail. We demonstrate how to use this knowledge to refine these methods and improve their predictive capabilities. This method provides a reliable base upon which to improve the design of biological systems. PMID:26379153

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

    PubMed

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

    2015-06-01

    For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80% can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins. PMID:25929326

  13. Biosynthetic Studies of 13-Desmethylspirolide C Produced by Alexandrium ostenfeldii (= A. peruvianum): Rationalization of the Biosynthetic Pathway Following Incorporation of (13)C-Labeled Methionine and Application of the Odd-Even Rule of Methylation.

    PubMed

    Anttila, Matthew; Strangman, Wendy; York, Robert; Tomas, Carmelo; Wright, Jeffrey L C

    2016-03-25

    Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding (13)C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd-even methylation rule, the complete biosynthetic pathway has been established.

  14. Labeling strategies for 13C-detected aligned-sample solid-state NMR of proteins

    NASA Astrophysics Data System (ADS)

    Filipp, Fabian V.; Sinha, Neeraj; Jairam, Lena; Bradley, Joel; Opella, Stanley J.

    2009-12-01

    13C-detected solid-state NMR experiments have substantially higher sensitivity than the corresponding 15N-detected experiments on stationary, aligned samples of isotopically labeled proteins. Several methods for tailoring the isotopic labeling are described that result in spatially isolated 13C sites so that dipole-dipole couplings among the 13C are minimized, thus eliminating the need for homonuclear 13C- 13C decoupling in either indirect or direct dimensions of one- or multi-dimensional NMR experiments that employ 13C detection. The optimal percentage for random fractional 13C labeling is between 25% and 35%. Specifically labeled glycerol and glucose can be used at the carbon sources to tailor the isotopic labeling, and the choice depends on the resonances of interest for a particular study. For investigations of the protein backbone, growth of the bacteria on [2- 13C]-glucose-containing media was found to be most effective.

  15. Enhancing Phospholipid Fatty Acid Profiling of Soil Bacterial Communities via Substrate- Specific 13C-labelling

    NASA Astrophysics Data System (ADS)

    Evershed, R. P.; Maxfield, P. J.; Bingham, E. M.; Dildar, N.; Brennand, E. L.; Hornibrook, E.

    2008-12-01

    A range of culture-independent methods, has recently emerged to study environmental microorganisms in situ[1]. One such method is phospholipid fatty acid (PLFA) analysis, wherein these ubiquitous membrane lipids provide a powerful tool for the study of unculturable soil microorganisms. PLFA analyses have been used to investigate the impacts of a wide range of environmental factors on the soil microbial community. An acknowledged shortcoming of the PLFAs approach is the lack the chemotaxonoic specificity, which restricts the ability of the method to probe the activities of specific functional groups of the microbial community selectively. However, the selectivity of PLFAs analyses can be enhanced by incubating soils with 13C- labelled substrates followed by gas chromatography-combustion-isotope ratio mass spectrometry to reveal the specific PLFAs incorporating the 13C-label. The application of this approach will be demonstrated through our recent work on methanotrophic bacteria in soils. We applied this approach initially to mineral soils[2] and then extended chemotaxonomic assessments by using a combination of 13C-labelled PLFAs and hopanoids [3]. We have used this approach to explore the properties of high affinity methanotrophs in a range of environments, investigating the relationship between methane oxidation rates and the nature and magnitude of the methanotrophic community for the first time[4,5] More recently we extended the technique using a novel time series 13C-labelling of PLFAs[6] to estimate the rate and progression of 13C- label incorporation and turnover of methanotrophic populations. This modified approach has been used to investigate the impacts of various environmental variables, e.g. soil type, vegetation cover and land use, on the methanotrophic biomass[7.8]. The unique nature of the 13CH4 as a gaseous substate/carbon source means that can be readily introduced into soils via a specific subset of the soil microbial biomass, thereby offering many

  16. 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

  17. Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo.

    PubMed Central

    Berthold, H K; Hachey, D L; Reeds, P J; Thomas, O P; Hoeksema, S; Klein, P D

    1991-01-01

    The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo. Images PMID:11607211

  18. Synthesis of isotopically labeled R- or S-[.sup.13C, .sup.2H] glycerols

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2008-01-22

    The present invention is directed to asymmetric chiral labeled glycerols including at least one chiral atom, from one to two .sup.13C atoms and from zero to four deuterium atoms bonded directly to a carbon atom, e.g., (2S) [1,2-.sup.13C.sub.2]glycerol and (2R) [1,2-.sup.13C.sub.2]glycerol, and to the use of such chiral glycerols in the preparation of labeled amino acids.

  19. Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain.

    PubMed

    Kamiya, Yukiko; Yamamoto, Sayoko; Chiba, Yasunori; Jigami, Yoshifumi; Kato, Koichi

    2011-08-01

    This report describes a novel method for overexpression of (13)C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly (13)C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man(8)GlcNAc(2) oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, (13)C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific (13)C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The (13)C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.

  20. Interaction between rhizosphere microorganisms and plant roots: 13C fluxes in the rhizosphere after pulse labeling

    NASA Astrophysics Data System (ADS)

    Yevdokimov, I. V.; Ruser, R.; Buegger, F.; Marx, M.; Munch, J. C.

    2007-07-01

    The input dynamics of labeled C into pools of soil organic matter and CO2 fluxes from soil were studied in a pot experiment with the pulse labeling of oats and corn under a 13CO2 atmosphere, and the contribution of the root and microbial respiration to the emission of CO2 from the soil was determined from the fluxes of labeled C in the microbial biomass and the evolved carbon dioxide. A considerable amount of 13C (up to 96% of the total amount of the label found in the rhizosphere soil) was incorporated into the biomass of the rhizosphere microorganisms. The diurnal fluctuations of the labeled C pools in the microbial biomass, dissolved organic carbon, and CO2 released in the rhizosphere of oats and corn were related to the day/night changes, i.e., to the on and off periods of the photosynthetic activity of the plants. The average contribution of the corn root respiration (70% of the total CO2 emission from the soil surface) was higher than that of the oats roots (44%), which was related to the lower incorporation of rhizodeposit carbon into the microbial biomass in the soil under the corn plants than in the soil under the oats plants.

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

    DOEpatents

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

    2001-01-01

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

  2. 13C Incorporation into Signature Fatty Acids as an Assay for Carbon Allocation in Arbuscular Mycorrhiza

    PubMed Central

    Olsson, Pål Axel; van Aarle, Ingrid M.; Gavito, Mayra E.; Bengtson, Per; Bengtsson, Göran

    2005-01-01

    The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1ω5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating 13C enrichment of 16:1ω5 and compared it with 13C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [13C]glucose. The 13C enrichment of neutral lipid fatty acid 16:1ω5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for 13C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1ω5 than for the root specific neutral lipid fatty acid 18:2ω6,9. We labeled plant assimilates by using 13CO2 in whole-plant experiments. The extraradical mycelium often was more enriched for 13C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between 13C enrichment in neutral lipid fatty acid 16:1ω5 and total 13C in extraradical mycelia in different systems (r2 = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the 13C enrichment of 16:1ω5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia. PMID:15870350

  3. Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination

    PubMed Central

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2014-01-01

    Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from 13C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of 13C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the 13C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the 13C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in 13C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in 13C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions. PMID:23828762

  4. Economical synthesis of 13C-labeled opiates, cocaine derivatives and selected urinary metabolites by derivatization of the natural products.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2015-01-01

    The illegal use of opiates and cocaine is a challenge world-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the active ingredients and their metabolites are needed both for controlling administration in the clinic and to detect drugs of abuse. Especially, (13)C-labeled compounds are useful for identification and quantification purposes by mass spectroscopic techniques, potentially increasing accuracy by minimizing ion alteration/suppression effects. Thus, the synthesis of [acetyl-(13)C4]heroin, [acetyl-(13)C4-methyl-(13)C]heroin, [acetyl-(13)C2-methyl-(13)C]6-acetylmorphine, [N-methyl-(13)C-O-metyl-(13)C]codeine and phenyl-(13)C6-labeled derivatives of cocaine, benzoylecgonine, norcocaine and cocaethylene was undertaken to provide such reference materials. The synthetic work has focused on identifying (13)C atom-efficient routes towards these derivatives. Therefore, the (13)C-labeled opiates and cocaine derivatives were made from the corresponding natural products.

  5. Characterization of uniformly and atom-specifically 13C-labeled heparin and heparan sulfate polysaccharide precursors using 13C NMR spectroscopy and ESI mass spectrometry

    PubMed Central

    Nguyen, Thao K. N.; Tran, Vy M.; Victor, Xylophone V.; Skalicky, Jack J.; Kuberan, Balagurunathan

    2010-01-01

    The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin/heparan sulfate oligosaccharide or disaccharide fragments using simple 1D 1H NMR spectroscopy is often complicated by the extensive signal overlap. 13C NMR signals, on the other hand, overlap less and therefore, 13C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific 13C-labeled heparin/heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly 13C- and 15N-labeled precursors were also produced and characterized by 13C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of 13C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% 13C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome–proteome interactions. PMID:20832774

  6. The biosynthetic pathway of curcuminoid in turmeric (Curcuma longa) as revealed by 13C-labeled precursors.

    PubMed

    Kita, Tomoko; Imai, Shinsuke; Sawada, Hiroshi; Kumagai, Hidehiko; Seto, Haruo

    2008-07-01

    In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid > ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

  7. A roadmap for interpreting 13C metabolite labeling patterns from cells

    PubMed Central

    Buescher, Joerg M.; Antoniewicz, Maciek R.; Boros, Laszlo G.; Burgess, Shawn C.; Brunengraber, Henri; Clish, Clary B.; DeBerardinis, Ralph J.; Feron, Olivier; Frezza, Christian; Ghesquiere, Bart; Gottlieb, Eyal; Hiller, Karsten; Jones, Russell G.; Kamphorst, Jurre J.; Kibbey, Richard G.; Kimmelman, Alec C.; Locasale, Jason W.; Lunt, Sophia Y.; Maddocks, Oliver D. K.; Malloy, Craig; Metallo, Christian M.; Meuillet, Emmanuelle J.; Munger, Joshua; Nöh, Katharina; Rabinowitz, Joshua D.; Ralser, Markus; Sauer, Uwe; Stephanopoulos, Gregory; St-Pierre, Julie; Tennant, Daniel A.; Wittmann, Christoph; Vander Heiden, Matthew G.; Vazquez, Alexei; Vousden, Karen; Young, Jamey D.; Zamboni, Nicola; Fendt, Sarah-Maria

    2015-01-01

    Measuring intracellular metabolism has increasingly led to important insights in biomedical research. 13C tracer analysis, although less information-rich than quantitative 13C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting 13C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. PMID:25731751

  8. Using Position-Specific 13C and 14C Labeling and 13C-PLFA Analysis to Assess Microbial Transformations of Free Versus Sorbed Alanine

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Herschbach, J.; Bore, E. K.; Kuzyakov, Y.; Dippold, M. A.

    2015-12-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model substance for the pool of LMWOS. To assess transformations of sorbed alanine, we added position-specific and uniformly 13C and 14C labeled alanine tracer to soil that had previously been sterilized by γ-radiation. The labeled soil was added to non-sterilized soil from the same site and incubated. Soil labeled with the same tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time intervals. The incorporation of 14C into microbial biomass was determined by chloroform fumigation extraction (CFE), and utilization of individual C positions by distinct microbial groups was evaluated by 13C-phospholipid fatty acid analysis (PLFA). A dual peak in the respired CO2 revealed two sorption mechanisms. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we applied the divergence index (DI). The DI reveals the convergent or divergent behavior of C from individual molecule positions during microbial utilization. Alanine C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFA. This indicates that sorption by the COOH group does not protect this group from preferential oxidation. Microbial metabolism was determinative for the preferential oxidation of individual molecule positions. The use of position-specific labeling revealed mechanisms and kinetics of microbial utilization of sorbed and non

  9. The fate of (13)C-labelled and non-labelled inulin predisposed to large bowel fermentation in rats.

    PubMed

    Butts, Christine A; Paturi, Gunaranjan; Tavendale, Michael H; Hedderley, Duncan; Stoklosinski, Halina M; Herath, Thanuja D; Rosendale, Douglas; Roy, Nicole C; Monro, John A; Ansell, Juliet

    2016-04-01

    The fate of stable-isotope (13)C labelled and non-labelled inulin catabolism by the gut microbiota was assessed in a healthy rat model. Sprague-Dawley male rats were randomly assigned to diets containing either cellulose or inulin, and were fed these diets for 3 days. On day (d) 4, rats allocated to the inulin diet received (13)C-labelled inulin. The rats were then fed the respective non-labelled diets (cellulose or inulin) until sampling (d4, d5, d6, d7, d10 and d11). Post feeding of (13)C-labelled substrate, breath analysis showed that (13)C-inulin cleared from the host within a period of 36 hours. Faecal (13)C demonstrated the clearance of inulin from gut with a (13)C excess reaching maximum at 24 hours (d5) and then declining gradually. There were greater variations in caecal organic acid concentrations from d4 to d6, with higher concentrations of acetic, butyric and propionic acids observed in the rats fed inulin compared to those fed cellulose. Inulin influenced caecal microbial glycosidase activity, increased colon crypt depth, and decreased the faecal output and polysaccharide content compared to the cellulose diet. In summary, the presence of inulin in the diet positively influenced large bowel microbial fermentation.

  10. Measurement of multiple psi torsion angles in uniformly 13C,15N-labeled alpha-spectrin SH3 domain using 3D 15N-13C-13C-15N MAS dipolar-chemical shift correlation spectroscopy.

    PubMed

    Ladizhansky, Vladimir; Jaroniec, Christopher P; Diehl, Annette; Oschkinat, Hartmut; Griffin, Robert G

    2003-06-01

    We demonstrate the simultaneous measurement of several backbone torsion angles psi in the uniformly (13)C,(15)N-labeled alpha-Spectrin SH3 domain using two different 3D 15N-13C-13C-15N dipolar-chemical shift magic-angle spinning (MAS) NMR experiments. The first NCCN experiment utilizes double quantum (DQ) spectroscopy combined with the INADEQUATE type 13C-13C chemical shift correlation. The decay of the DQ coherences formed between 13C'(i) and 13C(alphai) spin pairs is determined by the "correlated" dipolar field due to 15N(i)-13C(alphai) and 13C'(i)-15N(i+1) dipolar couplings and is particularly sensitive to variations of the torsion angle in the regime |psi| > 140 degrees. However, the ability of this experiment to constrain multiple psi-torsion angles is limited by the resolution of the 13C(alpha)-(13)CO correlation spectrum. This problem is partially addressed in the second approach described here, which is an NCOCA NCCN experiment. In this case the resolution is enhanced by the superior spectral dispersion of the 15N resonances present in the 15N(i+1)-13C(alphai) part of the NCOCA chemical shift correlation spectrum. For the case of the 62-residue alpha-spectrin SH3 domain, we determined 13 psi angle constraints with the INADEQUATE NCCN experiment and 22 psi constraints were measured in the NCOCA NCCN experiment.

  11. Utilization of low molecular weight organics by soil microorganisms: combination of 13C-labelling with PLFA analysis

    NASA Astrophysics Data System (ADS)

    Gunina, Anna; Dippold, Michaela; Kuzyakov, Yakov

    2014-05-01

    Microbial metabolisation is the main transformation pathway of low molecular weight organic substances (LMWOS), but detailed knowledge concerning the fate of LMWOS in soils is strongly limited. Considering that various LMWOS classes enter biochemical cycles at different steps, we hypothesise that the percentage of their LMWOS-Carbon (C) used for microbial biomass (MB) production and consequently medium-term stabilisation in soil is different. We traced the three main groups of LMWOS: amino acids, sugars and carboxylic acids, by uniformly labelled 13C-alanine, -glutamate, -glucose, -ribose, -acetate and -palmitate. Incorporation of 13C from these LMWOS into MB (fumigation-extraction method) and into phospholipid fatty acids (PLFAs) (Bligh-Dyer extraction, purification and GC-C-IRMS measurement) was investigated under field conditions 3 d and 10 d after LMWOS application. The activity of microbial utilization of LMWOS for cell membrane construction was estimated by replacement of PLFA-C with 13C. Decomposition of LMWOS-C comprised 20-65% of the total label, whereas incorporation of 13C into MB amounted to 20-50% of initially applied 13C on day three and was reduced to 5-30% on day 10. Incorporation of 13C-labelled LMWOS into MB followed the trend sugars > carboxylic acids > amino acids. Differences in microbial utilisation between LMWOS were observed mainly at day 10. Thus, instead of initial rapid uptake, further metabolism within microbial cells accounts for the individual fate of C from different LMWOS in soils. Incorporation of 13C from each LMWOS into each PLFA occurred, which reflects the ubiquitous ability of all functional microbial groups for LMWOS utilization. The preferential incorporation of palmitate can be attributed to its role as a direct precursor for many fatty acids (FAs) and PLFA formation. Higher incorporation of alanine and glucose compared to glutamate, ribose and acetate reflect the preferential use of glycolysis-derived substances in the FAs

  12. Delayed labelling of brain glutamate after an intra-arterial [13C]glucose bolus: evidence for aerobic metabolism of guinea pig brain glycogen store.

    PubMed

    Griffin, J L; Rae, C; Radda, G K; Matthews, P M

    1999-07-01

    Glycogen in glial cells is the largest store of glucose equivalents in the brain. Here we describe evidence that brain glycogen contributes to aerobic energy metabolism of the guinea pig brain in vivo. Five min after an intra-arterial bolus injection of d-[U-14C]glucose, 28+/-11% of the radioactivity in brain tissue was associated with the glycogen fraction, indicating that a significant proportion of labelled glucose taken up by the brain is converted to glycogen shortly after bolus infusion. Incorporation of 13C-label into lactate generated by brains made ischaemic after d-[1-13C]glucose injection confirms that these glucose equivalents can be mobilised for anaerobic glucose metabolism. Aerobic metabolism was monitored by following the time course of 13C-incorporation into glutamate in guinea pig cortex and cerebellum in vivo. After an intra-arterial bolus injection of d-[1-13C]glucose, glutamate labelling reached a maximum 40-60 min after injection, suggesting that a slowly metabolised pool of labelled glucose equivalents was present. As the concentration of 13C-labelled glucose in blood was shown to decrease below detectable levels within 5 min of bolus injection, this late phase of glutamate labelling must occur with mobilisation of a brain storage pool of labelled glucose equivalents. We interpret this as evidence that glucose equivalents in glycogen may contribute to energy metabolism in the aerobic guinea pig brain.

  13. Accurate measurements of {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy

    SciTech Connect

    Straasø, Lasse Arnt; Nielsen, Jakob Toudahl; Bjerring, Morten; Nielsen, Niels Chr.; Khaneja, Navin

    2014-09-21

    Application of sets of {sup 13}C-{sup 13}C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ({sup 13}C′) and aliphatic ({sup 13}C{sub aliphatic}) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly {sup 13}C,{sup 15}N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of {sup 13}C′-{sup 13}C{sub aliphatic} distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform {sup 13}C,{sup 15}N-labeling on the FGAIL fragment.

  14. 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

  15. Synthesis and applications of selectively {sup 13}C-labeled RNA

    SciTech Connect

    SantaLucia, J. Jr.; Shen, L.X.; Lewis, H.; Cai, Z.; Tinoci, I. Jr.

    1994-12-01

    Spectral overlap is a substantial problem in NMR studies of RNA molecules >30 nucleotides. To overcome this difficulty, we synthesized selectively {sup 13}C-labeled RNAs and adapted several isotope-edited two- and three-dimensional NMR experiments originally developed for protein studies. We optimized protocols for synthesis of multi-gram quantities of CTP, UTp, ATP, and GTP using a combination of synthetic organic and enzymatic methods. Uracil is prepared in 40 to 50% yield from {sup 13}C-cyanide in two steps. Using acetyl- tribenzoyl-ribose and standard chemistry uracil is then attached to the sugar (90% yield). The tribenzoyl-uridine intermediate is converted into uridine or cytidine quantitatively, depending on the deblocking protocol. Labeled purines are synthesized using simple pyrimidine precursors and reacting with {sup 13}C-formic acid (80% yield). Purine nucleosides are then synthesized using uridine phosphorylase and purine nucleoside phosphorylase. The nucleosides were converted to NMPs by treatment with POC1{sub 3} in triethylphosphate. We converted NMPs to NTPs by standard enzymatic methods. Selectively labeled RNAs were synthesized by run-off transcription using {sup 13}C-labeled NTPs. Several different strategies help solve over-lap problems in larger RNAs. Isotope-edited two-dimensional NMR experiments such as {omega}1-1/2 X-filtered NOESY simplify NMR spectra by dividing the normal NOESY spectrum into two subspectra-one involving NOEs from protons bound to {sup 12}C and one from protons bound to {sup 13}C. For example, we labeled A and U residues of a 34-nucleotide pseudoknot, and the {sup 12}C subspectrum of the 1/2 X-filtered NOESY contained NOEs only from G and C residues (along with adenine 2H); the {sup 13}C subspectrum contained NOEs only from A and U residues. Each subspectrum has less overlap than the NOESY of an unlabeled sample; the editing strategy allows each resonance to be identified by residue type (A, C, G, or U).

  16. Assessing microbial utilization of free versus sorbed Alanine by using position-specific 13C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Herschbach, Jennifer; Apostel, Carolin; Spielvogel, Sandra; Kuzyakov, Yakov; Dippold, Michaela

    2016-04-01

    Microbial utilization is a key transformation process of soil organic matter (SOM). Sorption of low molecular weight organic substances (LMWOS) to soil mineral surfaces blocks or delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil science, combined with 13C-phospholipid fatty acid (PLFA) analysis, to assess microbial utilization of sorbed and non-sorbed Alanine in soil. Alanine has various functional groups enabling different sorption mechanisms via its positive charge (e.g. to clay minerals by cation exchange), as well as via its negative charge (e.g. to iron oxides by ligand exchange). To assess changes in the transformation pathways caused by sorption, we added uniformly and position-specifically 13C and 14C labeled Alanine to the Ap of a loamy Luvisol in a short-term (10 days) incubation experiment. To allow for sorption of the tracer solution to an aliquot of this soil, microbial activity was minimized in this subsample by sterilizing the soil by γ-radiation. After shaking, the remaining solutions were filtered and the non-sorbed Alanine was removed with Millipore water and then added to non-sterilized soil. For the free Alanine treatment, solutions with Alanine of similar amount and isotopic composition were prepared, added to the soil and incubated as well. The respired CO2 was trapped in NaOH and its 14C-activity was determined at increasing times intervals. Microbial utilization of Alanine's individual C positions was evaluated in distinct microbial groups classified by 13C-PLFA analysis. Sorption to soil minerals delayed respiration to CO2 and reduced initial respiration rate by 80%. Irrespective of sorption, the highest amount was respired from the carboxylic position (C-1), whereas the amino-bound (C-2) and the methylic position (C-3) were preferentially incorporated into PLFA of microorganisms due to the

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

    PubMed Central

    Sinha, Neeraj; Grant, Christopher V.; Park, Sang Ho; Brown, Jonathan Miles; Opella, Stanley J.

    2013-01-01

    Initial steps in the development of a suite of triple-resonance 1H/13C/15N solid-state NMR experiments applicable to aligned samples of 13C and 15N labeled proteins are described. The experiments take advantage of the opportunities for 13C detection without the need for homonuclear 13C/13C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are ~20% randomly labeled with 13C in all backbone and side chain carbon sites and ~100% uniformly 15N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are 13C labeled at only the α-carbon and 15N labeled at the amide nitrogen of a few residues. The requirement for homonuclear 13C/13C decoupling while detecting 13C signals is avoided in the first case because of the low probability of any two 13C nuclei being bonded to each other; in the second case, the labeled 13Cα sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the 13C chemical shift and 1H–13C and 15N–13C heteronuclear dipolar coupling frequencies associated with the 13Cα and 13C′ backbone sites, which provide orientation constraints complementary to those derived from the 15N labeled amide backbone sites. 13C/13C spin-exchange experiments identify proximate carbon sites. The ability to measure 13C–15N dipolar coupling frequencies and correlate 13C and 15N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the 13C chemical shift and 1H–13C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach. PMID:17293139

  18. The Fate of Oral Glucosamine Traced by 13C Labeling in the Dog

    PubMed Central

    Dodge, George R.; Regatte, Ravinder R.; Noyszewski, Elizabeth A.; Hall, Jeffery O.; Sharma, Akella V.; Callaway, D. Allen; Reddy, Ravinder

    2011-01-01

    Objective: It has remained ambiguous as to whether oral dosing of glucosamine (GlcN) would make its way to the joint and affect changes in the cartilage, particularly the integrity of cartilage and chondrocyte function. The objective of this study was to trace the fate of orally dosed GlcN and determine definitively if GlcN was incorporated into cartilage proteoglycans. Design: Two dogs were treated with 13C-GlcN-HCl by oral dosing (500 mg/dog/d for 2 weeks and 250 mg/dog/d for 3 weeks). Cartilage was harvested from the tibial plateau and femoral condyles along with tissue specimens from the liver, spleen, heart, kidney, skin, skeletal muscle, lung, and costal cartilage. Percentages of 13C and 13C-GlcN present in each tissue sample were determined by inductively coupled plasma mass spectroscopy (ICP-MS) and nuclear magnetic resonance spectroscopy, respectively. Results: In the case of dog 1 (2-week treatment), there was an increase of 2.3% of 13C present in the articular cartilage compared to the control and an increase of 1.6% of 13C in dog 2 compared to control. As to be expected, the highest percentage of 13C in the other tissues tested was found in the liver, and the remaining tissues had percentages of 13C less than that of articular cartilage. Conclusion: The results are definitive and for the first time provide conclusive evidence that orally given GlcN can make its way through the digestive tract and be used by chondrocytes in joint cartilage, thereby potentially having an effect on the available GlcN for proteoglycan biosynthesis. PMID:26069586

  19. Anaerobic Methane Oxidation in Soils - revealed using 13C-labelled methane tracers

    NASA Astrophysics Data System (ADS)

    Riekie, G. J.; Baggs, E. M.; Killham, K. S.; Smith, J. U.

    2008-12-01

    In marine sediments, anaerobic methane oxidation is a significant biogeochemical process limiting methane flux from ocean to atmosphere. To date, evidence for anaerobic methane oxidation in terrestrial environments has proved elusive, and its significance is uncertain. In this study, an isotope dilution method specifically designed to detect the process of anaerobic methane oxidation in methanogenic wetland soils is applied. Methane emissions of soils from three contrasting permanently waterlogged sites in Scotland are investigated in strictly anoxic microcosms to which 13C- labelled methane is added, and changes in the concentration and 12C/13C isotope ratios of methane and carbon dioxide are subsequently measured and used to calculate separate the separate components of the methane flux. The method used takes into account the 13C-methane associated with methanogenesis, and the amount of methane dissolved in the soil. The calculations make no prior assumptions about the kinetics of methane production or oxidation. The results indicate that methane oxidation can take place in anoxic soil environments. The clearest evidence for anaerobic methane oxidation is provided by soils from a minerotrophic fen site (pH 6.0) in Bin Forest underlain by ultra-basic and serpentine till. In the fresh soil anoxic microcosms, net consumption methane was observed, and the amount of headspace 13C-CO2 increased at a greater rate than the 12+13C-CO2, further proof of methane oxidation. A net increase in methane was measured in microcosms of soil from Murder Moss, an alkaline site, pH 6.5, with a strong calcareous influence. However, the 13C-CH4 data provided evidence of methane oxidation, both in the disappearance of C- CH4 and appearance of smaller quantities of 13C-CO2. The least alkaline (pH 5.5) microcosms, of Gateside Farm soil - a granitic till - exhibited net methanogenesis and the changes in 13C-CH4 and 13C-CO2 here followed the pattern expected if no methane is consumed

  20. 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

  1. Differential growth of the fungus Absidia cylindrospora on 13C/15N-labelled media.

    PubMed

    Crotty, F V; Blackshaw, R P; Murray, P J

    2011-06-15

    Many studies utilise enrichment of stable isotopes as tracers to follow the interactions occurring within soil food webs and methods have been developed to enrich bacteria, soil fauna and plant litter, Here for the first time we attempt to enrich a soil fungus to 99 atom% with (13)C and (15)N stable isotopes. In this study our objectives were to (a) assess whether the saprotrophic zygomycete fungus Absidia cylindrospora could grow on a medium enriched to 99 atom% with (13)C-glucose and (15)N-ammonium chloride, (b) to determine the level of enrichment obtained, and (c) to examine the change in growth rate of this fungus while it was growing on the dually enriched medium. To achieve this, the fungus was grown on agar enriched with (13)C and (15)N to 99 atom% and its growth rate monitored. The results showed that A. cylindrospora would grow on the highly labelled growth medium, but that its rate of growth was affected compared with the rate on either natural abundance media or media highly enriched with a single isotope ((13)C or (15)N). The implications of these results is that although the fungus is able to utilise these heavier isotopes, the biochemical processes involved in growth are affected, and consideration should be given to these differences when using stable isotope tracers in, for example, soil food web studies.

  2. Economical synthesis of 13C-labeled opiates, cocaine derivatives and selected urinary metabolites by derivatization of the natural products.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2015-01-01

    The illegal use of opiates and cocaine is a challenge world-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the active ingredients and their metabolites are needed both for controlling administration in the clinic and to detect drugs of abuse. Especially, (13)C-labeled compounds are useful for identification and quantification purposes by mass spectroscopic techniques, potentially increasing accuracy by minimizing ion alteration/suppression effects. Thus, the synthesis of [acetyl-(13)C4]heroin, [acetyl-(13)C4-methyl-(13)C]heroin, [acetyl-(13)C2-methyl-(13)C]6-acetylmorphine, [N-methyl-(13)C-O-metyl-(13)C]codeine and phenyl-(13)C6-labeled derivatives of cocaine, benzoylecgonine, norcocaine and cocaethylene was undertaken to provide such reference materials. The synthetic work has focused on identifying (13)C atom-efficient routes towards these derivatives. Therefore, the (13)C-labeled opiates and cocaine derivatives were made from the corresponding natural products. PMID:25816077

  3. 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

  4. Methanogenic capabilities of ANME-archaea deduced from (13) C-labelling approaches.

    PubMed

    Bertram, Sebastian; Blumenberg, Martin; Michaelis, Walter; Siegert, Michael; Krüger, Martin; Seifert, Richard

    2013-08-01

    Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME-1 or ANME-2. The (13) C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate-dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME-1 and ANME-2. Considerable methane production rates (MPRs) from CO2 -reduction were observed, particularly from ANME-2 dominated samples and in the presence of methane, which supports the hypothesis of a co-occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through (13) C-assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the (13) C-uptake into lipids, ANME-1 was found to thrive also under methane free conditions. Finally, C35 -isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME-1, most likely functioning as a hydrogen sink during methanogenesis.

  5. Determination of the Orientation and Dynamics of Ergosterol in Model Membranes Using Uniform 13C Labeling and Dynamically Averaged 13C Chemical Shift Anisotropies as Experimental Restraints

    PubMed Central

    Soubias, O.; Jolibois, F.; Massou, S.; Milon, A.; Réat, V.

    2005-01-01

    A new strategy was established to determine the average orientation and dynamics of ergosterol in dimyristoylphosphatidylcholine model membranes. It is based on the analysis of chemical shift anisotropies (CSAs) averaged by the molecular dynamics. Static 13C CSA tensors were computed by quantum chemistry, using the gauge-including atomic-orbital approach within Hartree-Fock theory. Uniformly 13C-labeled ergosterol was purified from Pichia pastoris cells grown on labeled methanol. After reconstitution into dimyristoylphosphatidylcholine lipids, the complete 1H and 13C assignment of ergosterol's resonances was performed using a combination of magic-angle spinning two-dimensional experiments. Dynamically averaged CSAs were determined by standard side-band intensity analysis for isolated 13C resonances (C3 and ethylenic carbons) and by off-magic-angle spinning experiments for other carbons. A set of 18 constraints was thus obtained, from which the sterol's molecular order parameter and average orientation could be precisely defined. The validity of using computed CSAs in this strategy was verified on cholesterol model systems. This new method allowed us to quantify ergosterol's dynamics at three molar ratios: 16 mol % (Ld phase), 30 mol % (Lo phase), and 23 mol % (mixed phases). Contrary to cholesterol, ergosterol's molecular diffusion axis makes an important angle (14°) with the inertial axis of the rigid four-ring system. PMID:15923221

  6. Computational Platform for Flux Analysis Using 13C-Label Tracing- Phase I SBIR Final Report

    SciTech Connect

    Van Dien, Stephen J.

    2005-04-12

    Isotopic label tracing is a powerful experimental technique that can be combined with metabolic models to quantify metabolic fluxes in an organism under a particular set of growth conditions. In this work we constructed a genome-scale metabolic model of Methylobacterium extorquens, a facultative methylotroph with potential application in the production of useful chemicals from methanol. A series of labeling experiments were performed using 13C-methanol, and the resulting distribution of labeled carbon in the proteinogenic amino acids was determined by mass spectrometry. Algorithms were developed to analyze this data in context of the metabolic model, yielding flux distributions for wild-type and several engineered strains of M. extorquens. These fluxes were compared to those predicted by model simulation alone, and also integrated with microarray data to give an improved understanding of the metabolic physiology of this organism.

  7. 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

  8. Investigations of enzymatic alterations of 2,4-dichlorophenol using {sup 13}C-nuclear magnetic resonance in combination with site-specific {sup 13}C-labeling: Understanding the environmental fate of this pollutant

    SciTech Connect

    Nanny, M.A.; Bortiatynski, J.M.; Tien, M.; Hatcher, P.G.

    1996-11-01

    The biodegradation of {sup 13}C-labeled 2,4-dichlorophenol (DCP labeled at the C-2 and C-6 positions), in the presence and absence of natural organic matter (NOM), by the white-rot fungus Phanerochaete chrysosporium, was examined using {sup 13}C-nuclear magnetic resonance (NMR). Using this method permitted the chemistry occurring at or near the labeled site to be followed. The formation of alkyl ethers and alkene ethers was observed. No aromatic by-products were detected, indicating that aromatic compounds are quickly degraded. Examining the reaction with time shows the exponential removal of 2,4-DCP and the consequential formation of labeled by-products, whose concentration reaches a maximum just before all 2,4-DCP is consumed. After this, the by-products degrade exponentially. The presence of NOM causes 2,4-DCP to be removed from the aqueous phase more quickly than in its absence and also causes the by-products to reach their maximum concentration much earlier. Degradation of the by-products occurs at a much greater rate in the presence of NOM. One hypothesis for this behavior is that the NOM interacts with 2,4-DCP and its by-products, allowing them to be incorporated into the fungal biomass. {sup 13}C-nuclear magnetic resonance spectra of the fungal biomass after NaOH extraction show the presence of alkanes and a small amount of 2,4-DCP.

  9. Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.

    PubMed

    Liu, Jing; Liu, Chang; Fan, Ying; Munro, Rachel A; Ladizhansky, Vladimir; Brown, Leonid S; Wang, Shenlin

    2016-05-01

    We demonstrate a novel sparse (13)C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically (13)C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

  10. Direct uptake of organic carbon by grass roots and allocation in leaves and phytoliths: 13C labeling evidence

    NASA Astrophysics Data System (ADS)

    Alexandre, A.; Balesdent, J.; Cazevieille, P.; Chevassus-Rosset, C.; Signoret, P.; Mazur, J.-C.; Harutyunyan, A.; Doelsch, E.; Basile-Doelsch, I.; Miche, H.; Santos, G. M.

    2015-12-01

    In the rhizosphere, the uptake of low molecular weight carbon (C) and nitrogen (N) by plant roots has been well documented. While organic N uptake relatively to total uptake is important, organic C uptake is supposed to be low relatively to the plant's C budget. Recently, radiocarbon analyses demonstrated that a fraction of C from the soil was occluded in amorphous silica micrometric particles that precipitate in plant cells (phytoliths). Here, we investigated whether and in which extent organic C absorbed by grass roots, under the form of either intact amino acids (AAs) or microbial metabolites, can feed the organic C occluded in phytoliths. For this purpose we added 13C- and 15N-labeled AAs to the silicon-rich hydroponic solution of the grass Festuca arundinacea. The experiment was designed to prevent C leakage from the labeled nutritive solution to the chamber atmosphere. After 14 days of growth, the 13C and 15N enrichments (13C-excess and 15N-excess) in the roots, stems and leaves, and phytoliths, as well as the 13C-excess in AAs extracted from roots and stems and leaves, were quantified relatively to a control experiment in which no labelled AAs were added. The net uptake of 13C derived from the labeled AAs supplied to the nutritive solution (AA-13C) by Festuca arundinacea represented 4.5 % of the total AA-13C supply. AA-13C fixed in the plant represented only 0.13 % of total C. However, the experimental conditions may have underestimated the extent of the process under natural and field conditions. Previous studies showed that 15N and 13C can be absorbed by the roots in several organic and inorganic forms. In the present experiment, the fact that phenylalanine and methionine, that were supplied in high amount to the nutritive solution, were more 13C-enriched than other AAs in the roots and stems and leaves strongly suggested that part of AA-13C was absorbed and translocated in its original AA form. The concentration of AA-13C represented only 0.15 % of the

  11. Monitoring CO[subscript 2] Fixation Using GC-MS Detection of a [superscript 13]C-Label

    ERIC Educational Resources Information Center

    Hammond, Daniel G.; Bridgham, April; Reichert, Kara; Magers, Martin

    2010-01-01

    Much of our understanding of metabolic pathways has resulted from the use of chemical and isotopic labels. In this experiment, a heavy isotope of carbon, [superscript 13]C, is used to label the product of the well-known RuBisCO enzymatic reaction. This is a key reaction in photosynthesis that converts inorganic carbon to organic carbon; a process…

  12. 13C NMR studies of gluconeogenesis in rat liver cells: Utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats

    PubMed Central

    Cohen, S. M.; Ogawa, S.; Shulman, R. G.

    1979-01-01

    The gluconeogenic pathway from [2-13C]glycerol and [1,3-13C]glycerol has been followed in suspensions of isolated rat hepatocytes at 25°C by 13C NMR at 90.5 MHz. The flow of label through the major pathway from glycerol to L-glycerol 3-phosphate and into glucose was followed in cells from control and triiodothyronine-treated rats. Treatment increased the rates of glucose formation and glycerol consumption 2-fold and decreased the αGP level to 40%. We calculate that ≈60% of the flux is through the mitochondrial glycerol phosphate dehydrogenase in cells from triiodothyronine-treated rats, compared with ≈15% in cells from the controls. Equal distribution of label between the trioses of glucose was obtained and, because the C3-C4 spin-spin coupling gives the distribution of labeled carbons in the same molecule, it was possible to measure the amount of triose from unlabeled fructose incorporated into the glucose labeled at carbons 1, 3, 4, and 6. About 10% of the hexoses had flowed through the pentose cycle and back into the hexose pathway in cells from fasted rats. From the distribution of label at glucose carbons not labeled via the major pathway and from the carbon spin-spin splitting patterns observed, we conclude that transketolase is reversible whereas transaldolase is essentially irreversible in the nonoxidative pentose branch. PMID:287001

  13. Synthesis of 13C and 15N labeled 2,4-dinitroanisole.

    PubMed

    Jagadish, Bhumasamudram; Field, Jim A; Chorover, Jon; Sierra-Alvarez, Reyes; Abrell, Leif; Mash, Eugene A

    2014-05-30

    Syntheses of [(13)C6]-2,4-dinitroanisole (ring-(13)C6) from [(13)C6]-anisole (ring-(13)C6) and [(15)N2]-2,4-dinitroanisole from anisole using in situ generated acetyl nitrate and [(15)N]-acetyl nitrate, respectively, are described. Treatment of [(13)C6]-anisole (ring-(13)C6) with acetyl nitrate generated in 100% HNO3 gave [(13)C6]-2,4-dinitroanisole (ring-(13)C6) in 83% yield. Treatment of anisole with [(15)N]-acetyl nitrate generated in 10 N [(15)N]-HNO3 gave [(15)N2 ]-2,4-dinitroanisole in 44% yield after two cycles of nitration. Byproducts in the latter reaction included [(15)N]-2-nitroanisole and [(15)N]-4-nitroanisole.

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

    PubMed Central

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

    2014-01-01

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

  15. 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

  16. 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

  17. Direct uptake of organically derived carbon by grass roots and allocation in leaves and phytoliths: 13C labeling evidence

    NASA Astrophysics Data System (ADS)

    Alexandre, Anne; Balesdent, Jérôme; Cazevieille, Patrick; Chevassus-Rosset, Claire; Signoret, Patrick; Mazur, Jean-Charles; Harutyunyan, Araks; Doelsch, Emmanuel; Basile-Doelsch, Isabelle; Miche, Hélène; Santos, Guaciara M.

    2016-03-01

    In the rhizosphere, the uptake of low-molecular-weight carbon (C) and nitrogen (N) by plant roots has been well documented. While organic N uptake relative to total uptake is important, organic C uptake is supposed to be low relative to the plant's C budget. Recently, radiocarbon analyses demonstrated that a fraction of C from the soil was occluded in amorphous silica micrometric particles that precipitate in plant cells (phytoliths). Here, we investigated whether and to what extent organically derived C absorbed by grass roots can feed the C occluded in phytoliths. For this purpose we added 13C- and 15N-labeled amino acids (AAs) to the silicon-rich hydroponic solution of the grass Festuca arundinacea. The experiment was designed to prevent C leakage from the labeled nutritive solution to the chamber atmosphere. After 14 days of growth, the 13C and 15N enrichments (13C excess and 15N excess) in the roots, stems and leaves as well as phytoliths were measured relative to a control experiment in which no labeled AAs were added. Additionally, the 13C excess was measured at the molecular level, in AAs extracted from roots and stems and leaves. The net uptake of labeled AA-derived 13C reached 4.5 % of the total AA 13C supply. The amount of AA-derived 13C fixed in the plant was minor but not nil (0.28 and 0.10 % of total C in roots and stems/leaves, respectively). Phenylalanine and methionine that were supplied in high amounts to the nutritive solution were more 13C-enriched than other AAs in the plant. This strongly suggested that part of AA-derived 13C was absorbed and translocated into the plant in its original AA form. In phytoliths, AA-derived 13C was detected. Its concentration was on the same order of magnitude as in bulk stems and leaves (0.15 % of the phytolith C). This finding strengthens the body of evidences showing that part of organic compounds occluded in phytoliths can be fed by C entering the plant through the roots. Although this experiment was done in

  18. Follow the Carbon: Laboratory Studies of 13C-Labeled Early Earth Haze Analogs

    NASA Astrophysics Data System (ADS)

    Hicks, R. K.; Day, D. A.; Mojzsis, S. J.; Jimenez, J. L.; Tolbert, M. A.

    2013-12-01

    While the Sun was still young and faint before the rise of molecular oxygen 2.4 Ga, early Earth might have been kept warm by an atmosphere containing the greenhouse gases methane and carbon dioxide in abundances greater than what is found on Earth today. It has been suggested that an atmosphere containing approximately 1000 ppmv methane and carbon dioxide could provided the needed greenhouse warming for liquid water to exist at the surface. Laboratory and modeling studies suggest that an atmosphere containing methane and carbon dioxide could lead to the formation of significant amounts of organic haze due to photochemical reactions initiated by Lyman-α (121.6 nm) excitation. Chemical mechanisms proposed to explain the chemistry rely on methane as the source of carbon in these hazes and treat carbon dioxide as a source of oxygen only. In the present work, we use isotopically labelled precursor gases to examine the source of carbon in photochemical haze formed in a CH4/CO2/N2 atmosphere. We generate haze analogs in the laboratory by far-UV irradiation of analog atmospheres containing permutations of 1,000 ppmv unlabeled and 13C-labeled methane and carbon. Products in the particle phase were analyzed by both unit mass resolution and high-resolution (m/Δm=5,000) aerosol mass spectrometry. Results indicate that carbon from carbon dioxide accounts for 20% (×5%) of the total carbon contained in the hazes. These results have implications for the geochemical interpretations of inclusions found in Archaean rocks on Earth, and for the astrobiological potential of other planetary atmospheres.

  19. {sup 13}C-enrichment at carbons 8 and 2 of uric acid after {sup 13}C-labeled folate dose in man

    SciTech Connect

    Baggott, Joseph E.; Gorman, Gregory S.; Morgan, Sarah L.; Tamura, Tsunenobu . E-mail: tamurat@uab.edu

    2007-09-21

    To evaluate folate-dependent carbon incorporation into the purine ring, we measured {sup 13}C-enrichment independently at C{sub 2} and C{sub 8} of urinary uric acid (the final catabolite of purines) in a healthy male after an independent oral dose of [6RS]-5-[{sup 13}C]-formyltetrahydrofolate ([6RS]-5-H{sup 13}CO-H{sub 4}folate) or 10-H{sup 13}CO-7,8-dihydrofolate (10-H{sup 13}CO-H{sub 2}folate). The C{sub 2} position was {sup 13}C-enriched more than C{sub 8} after [6RS]-5-H{sup 13}CO-H{sub 4}folate, and C{sub 2} was exclusively enriched after 10-H{sup 13}CO-H{sub 2}folate. The enrichment of C{sub 2} was greater from [6RS]-5-H{sup 13}CO-H{sub 4}folate than 10-H{sup 13}CO-H{sub 2}folate using equimolar bioactive doses. Our data suggest that formyl C of [6RS]-10-H{sup 13}CO-H{sub 4}folate was not equally utilized by glycinamide ribotide transformylase (enriches C{sub 8}) and aminoimidazolecarboxamide ribotide (AICAR) transformylase (enriches C{sub 2}), and the formyl C of 10-H{sup 13}CO-H{sub 2}folate was exclusively used by AICAR transformylase. 10-HCO-H{sub 2}folate may function in vivo as the predominant substrate for AICAR transformylase in humans.

  20. Stabilization of glucose-C in microbial cell membranes (PLFA) and cell walls (amino sugars) evaluated by 13C-labelling in a field experiment

    NASA Astrophysics Data System (ADS)

    Gunina, Anna; Kuzyakov, Yakov; Glaser, Bruno

    2015-04-01

    Microorganisms control carbon (C) cycle and strongly contribute to formation of soil organic matter. Strong differences in the turnover of microbial groups and cellular compounds complicate the assessment of their contribution to microbial food webs and C sequestration in soil in situ. The uptake and incorporation of 13C labeled glucose by microbial groups were traced during 50 days after the labeling under field conditions. 13C was analysed: i) in the cytosolic pool by chloroform fumigation extraction, ii) in cell membranes by phospholipid fatty acids (PLFA), iii) in cell walls by amino sugars, and iv) remaining in bulk soil. This allowed tracing C in microbial groups as well as cellular compounds. Mean residence times (MRT) of C in PLFA and the cytosol were 47 and 150 days, respectively. Such long cytosol MRT depends on its heterogeneous composition, which includes high and low molecular weight organics. Amino sugars were mainly originated from microbial residues and thus, observation periods higher than 1 year are required for estimation of their MRT. Relative 13C incorporation (13C portion in total pool C) was the highest for PLFAs (~1.5% at day 3), whereas 13C content of the cytosol and amino sugars was one and two orders of magnitude less, respectively. Relative 13C incorporation into amino sugars of living microorganisms showed only 0.57% on day 3. Therefore, the turnover of cell membrane components is two times faster than that of cell walls, even in living microorganisms. Both PLFAs and amino sugars showed that glucose C was preferentially used by bacteria. 13C incorporation into bacterial cell walls and membranes decreased with time, but increased or remained constant for fungi, reflecting faster turnover of bacteria than fungi. Consequently, bacteria contribute more to the decomposition of low molecular weight organics, whereas fungi consume bacterial products or necromass and contribute more to long-term C stabilisation. Thus, tracing of 13C in cellular

  1. Homonuclear dipolar recoupling techniques for structure determination in uniformly 13C-labeled proteins.

    PubMed

    Ladizhansky, Vladimir

    2009-11-01

    In solid-state NMR magic angle spinning is often used to remove line broadening associated with anisotropic interactions, such as chemical shift anisotropy and dipolar couplings. Dipolar recoupling refers to sequences of pulses designed to reintroduce dipolar interactions that are otherwise averaged by magic angle spinning. One of the key applications of homonuclear (and heteronuclear) dipolar recoupling is for the purpose of protein structure determination. Recoupling experiments, originally designed for applications in spin-pair labeled samples, have been revised in recent years for applications in samples with extensive or uniform incorporation of isotopic labels. In these samples multiple internuclear distances can in principle be probed simultaneously, but the dipolar truncation effects (i.e. attenuation of the effects of weak couplings by strong ones) circumvent such measurements. In this article we review some of the recent developments in homonuclear recoupling methods that allow overcoming this problem.

  2. Microbial utilization of sugars in soil assessed by position-specific labeling and compound-specific 13C-PLFA-analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Glaser, Bruno; Kuzyakov, Yakov

    2014-05-01

    For the transformation of low molecular weight organic substances (LMWOS) in soil, which is an important process in the turnover of organic matter, microbial utilization is one of the most important processes. Position-specific labeling combined with compound-specific 13C-PLFA-analysis allows a closer look on the mechanisms of LMWOS transformation in soil. We assessed short- (3 and 10 days) and long-term (half year) transformations of monosaccharides by adding position-specifically 13C labeled glucose and ribose to soil in a field experiment conducted on an agriculturally used luvisol located in north-western Bavaria. We quantified the microbial utilization of the different functional groups by 13C-analysis of microbial biomass with the chloroform-fumigation-extraction method (CFE). 13C-PLFA analysis enabled us to distinguish individual microbial groups and compare their C-utilization. Preferential degradation of glucoses C-3 and C-4 respectively C-1 position enabled differentiation between the two main hexose metabolic pathways - glycolysis and the pentose phosphate pathway. Microbial groups revealed different incorporation of specific C positions into their PLFA. The highest incorporation was reached by the prokaryotic gram- negative groups. The application of position-specifically labeled substances, coupled with compound-specific 13C-PLFA analysis opens a new way to investigate the microbial transformations of LMWOS in soil. Observing single C atoms and their utilization by specific microbial groups allow conclusions about the mechanisms and kinetics of microbial utilization and interaction between these groups and therefore will improve our understanding of soil carbon fluxes.

  3. Determination of sup 13 C labeling pattern of citric acid cycle intermediates by gas chromatography-mass spectrometry

    SciTech Connect

    Di Donato, L.; Montgomery, J.A.; Des Rosiers, C.; David, F.; Garneau, M.; Brunengraber, H. )

    1990-02-26

    Investigations of the regulation of the citric acid cycle require determination of labeling patterns of cycle intermediates. These were assayed to date, using infusion of: (i) ({sup 14}C)tracer followed by chemical degradation of intermediates and (ii) ({sup 13}C)tracer followed by NMR analysis of intermediates. The authors developed a strategy to analyze by GC-MS the ({sup 13}C) labeling pattern of {mu}mole samples of citrate (CIT), isocitrate (ICIT), 2-ketoglutarate (2-KG), glutamate (GLU) and glutamine (GLN). These are enzymatically or chemically converted to 2-KG, ICIT, 4-aminobutyrate (GABA) and 2-hydroxyglutarate (2-OHG). GC-MS analyses of TMS or TBDMS derivatives of these compounds yield the enrichment of each carbon. The authors confirmed the identity of each fragment using the spectra of (1-{sup 13}C), (5-{sup 13}C), (2,3,3,4,4-{sup 2}H{sub 5})glutamate and (1-{sup 13}C), (1,4-{sup 13}C)GABA.

  4. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    PubMed Central

    Sugai, Yoshinori; Ueno, Yohei; Hayashi, Ken-ichiro; Oogami, Shingo; Toyomasu, Tomonobu; Matsumoto, Sadamu; Natsume, Masahiro; Nozaki, Hiroshi; Kawaide, Hiroshi

    2011-01-01

    Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-13C6]mevalonate, all carbons were labeled with 13C stable isotope (>99%). The fully 13C-labeled product was subjected to 13C-13C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and

  5. [COMPARATIVE EVALUATION OF THE EFFECTIVENESS OF THE USE OF 13C-LABELED MIXED TRIGLYCERIDE AND 13C-STARCH BREATH TESTS IN PATIENTS WITH CHRONIC PANCREATITIS AFTER CHOLECYSTECTOMY].

    PubMed

    Sirchak, Ye S

    2015-01-01

    The results of a comprehensive study of 96 patients after cholecystectomy are provided. The higher sensitivity and informativeness of the 13C-labeled mixed triglyceride breath .test compared with 13C-starch breath test for determining functional pancreatic insufficiency in patients after cholecystectomy in early stages of its formation was set. PMID:27491156

  6. 13C-13C and 15N-13C correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: Amino acid-type assignments and evidence for multiple conformations

    NASA Astrophysics Data System (ADS)

    Bodner, Michele L.; Gabrys, Charles M.; Struppe, Jochem O.; Weliky, David P.

    2008-02-01

    Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an ˜20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform C13, N15 labeling over large contiguous regions of amino acids. Two-dimensional C13-C13 and N15-C13 spectra were obtained for the membrane-bound fusion peptides and an amino acid-type C13 assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the C13 chemical shifts were more consistent with β strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with β strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous C13 and N15 assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Cα nuclei. These sets of two shifts may indicate two β strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4T instrument, one C13-C13 correlation spectrum was obtained on a 16.4T instrument and was better resolved than the comparable 9.4T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.

  7. Complete assignment of the methionyl carbonyl carbon resonance in switch variant anti-dansyl antibodies labeled with (1- sup 13 C)methionine

    SciTech Connect

    Kato, Koichi; Matsunaga, C.; Igarashi, Takako; Kim, Hahyung; Odaka, Asano; Shimada, Ichio; Arata, Yoji )

    1991-01-01

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire C{sub H}1 domain is deleted. The switch variant antibodies were specifically labeled with (1-{sup 13}C)methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating {sup 15}N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of ({alpha}-{sup 15}N)lysine and ({sup 15}N)threonine, both of which cannot become the substrate of transaminases. It was found that {beta}-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with {sup 15}N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val. On the basis of the results of the present {sup 13}C study, possible use of the assigned carbonyl carbon resonances for the elucidation of the structure-function relationship in the antibody system has been briefly discussed.

  8. Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional (13)C labeling, two-dimensional [(13)C, (1)H] nuclear magnetic resonance, and comprehensive isotopomer balancing.

    PubMed

    Sriram, Ganesh; Fulton, D Bruce; Iyer, Vidya V; Peterson, Joan Marie; Zhou, Ruilian; Westgate, Mark E; Spalding, Martin H; Shanks, Jacqueline V

    2004-10-01

    Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-(13)C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [(13)C, (1)H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 (13)C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain sds. A high flux was found through the oxidative pentose phosphate pathway (19.99 +/- 4.39 micromol d(-1) cotyledon(-1), or 104.2 carbon mol +/- 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 +/- 5.00 micromol d(-1) cotyledon(-1), or 113.2 carbon mol +/-26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.

  9. Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe.

    PubMed

    Carpy, Alejandro; Patel, Avinash; Tay, Ye Dee; Hagan, Iain M; Macek, Boris

    2015-01-01

    Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, (13)C(6) (15)N(4)-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.

  10. Use of 13C-Labeled Substrates to Determine Relative Methane Production Rates in Hypersaline Microbial Communities

    NASA Astrophysics Data System (ADS)

    Kelley, C. A.; Bebout, B.; Chanton, J.

    2015-12-01

    Rates and pathways of methane production were determined from photosynthetic soft microbial mats and gypsum-encrusted endoevaporites collected in hypersaline environments from California, Mexico and Chile, as well as an organic-rich mud from a pond in the El Tatio volcanic fields, Chile. Samples (mud, homogenized soft mats and endoevaporites) were incubated anaerobically with deoxygenated site water, and the increase in methane concentration through time in the headspaces of the incubation vials was used to determine methane production rates. To ascertain the substrates used by the methanogens, 13C-labeled methylamines, methanol, dimethylsulfide, acetate or bicarbonate were added to the incubations (one substrate per vial) and the stable isotopic composition of the resulting methane was measured. The vials amended with 13C-labeled methylamines produced the most 13C-enriched methane, generally followed by the 13C-labeled methanol-amended vials. The stable isotope data and the methane production rates were used to determine first order rate constants for each of the substrates at each of the sites. Estimates of individual substrate use revealed that the methylamines produced 55 to 92% of the methane generated, while methanol was responsible for another 8 to 40%.

  11. Production and NMR signal optimization of hyperpolarized 13C-labeled amino acids

    NASA Astrophysics Data System (ADS)

    Parish, Christopher; Niedbalski, Peter; Ferguson, Sarah; Kiswandhi, Andhika; Lumata, Lloyd

    Amino acids are targeted nutrients for consumption by cancers to sustain their rapid growth and proliferation. 13C-enriched amino acids are important metabolic tracers for cancer diagnostics using nuclear magnetic resonance (NMR) spectroscopy. Despite this diagnostic potential, 13C NMR of amino acids however is hampered by the inherently low NMR sensitivity of the 13C nuclei. In this work, we have employed a physics technique known as dynamic nuclear polarization (DNP) to enhance the NMR signals of 13C-enriched amino acids. DNP works by transferring the high polarization of electrons to the nuclear spins via microwave irradiation at low temperature and high magnetic field. Using a fast dissolution method in which the frozen polarized samples are dissolved rapidly with superheated water, injectable solutions of 13C-amino acids with highly enhanced NMR signals (by at least 5,000-fold) were produced at room temperature. Factors that affect the NMR signal enhancement levels such as the choice of free radical polarizing agents and sample preparation will be discussed along with the thermal mixing physics model of DNP. The authors would like to acknowledge the support by US Dept of Defense Award No. W81XWH-14-1-0048 and Robert A. Welch Foundation Grant No. AT-1877.

  12. Multi-isotope labelling (13C, 18O, 2H) for studying organic matter cycling within plant-soil systems

    NASA Astrophysics Data System (ADS)

    Studer, M. S.; Abiven, S.; Schmidt, M. W. I.; Siegwolf, R. T. W.

    2012-04-01

    Carbon cycling has become of major interest for the understanding and mitigation of global climatic change. Terrestrial ecosystems have a large carbon sequestration potential, but many processes and fluxes of organic matter (OM) cycling within the plant-soil system are not yet well understood [1]. The dynamics of OM cycling within the plant soil-system are determined by environmental parameters, as well as chemical quality of OM input. A well-known technique to study OM dynamics is to label OM inputs with stable isotopes (e.g 13C). Changes in OM quality in the plant and in the soil can be assessed by compound specific isotopic analysis [2]. These techniques give a precise insight of the OM composition, but are laborious and expensive. Here we suggest a new multi-isotope labelling technique using stable 13C in combination with stable 18O and 2H isotopes, which provides information on OM quality by simple bulk material analysis. The method is based on the creation of an isotopic van Krevelen diagram, which is used to describe different compound groups by plotting the atomic ratios of O/C vs. H/C [3]. We could show that new assimilates can be labelled with 13C, 18O and 2H by adding the stable isotopes (continuously) in the gaseous phase (CO2 and water vapour) to the plants atmosphere. The label has been traced within the bulk material of different compartments of the plant-soil system (e.g. leaves, stems, roots, bulk soil). Our first results showed that after 2, 8 and 14 days of labelling the 18O/13C(new) ratio was notably different in leaf, stem and root tissue (0.0024, 0.0011 and 0.0007, respectively), suggesting a change in OM quality towards more C-rich compounds. d2H analysis will follow and an isotopic van Krevelen diagram will be produced (18O/13C(new) vs. 2H/13C(new)) to describe the changes in OM quality. The new multi-isotope labelling approach represent a powerful tool to address open questions in plant and soil research such as the allocation of organic

  13. Fate of xylem-transported 11C- and 13C-labeled CO2 in leaves of poplar.

    PubMed

    Bloemen, Jasper; Bauweraerts, Ingvar; De Vos, Filip; Vanhove, Christian; Vandenberghe, Stefaan; Boeckx, Pascal; Steppe, Kathy

    2015-04-01

    In recent studies, assimilation of xylem-transported CO2 has gained considerable attention as a means of recycling respired CO2 in trees. However, we still lack a clear and detailed picture on the magnitude of xylem-transported CO2 assimilation, in particular within leaf tissues. To this end, detached poplar leaves (Populus × canadensis Moench 'Robusta') were allowed to take up a dissolved (13)CO2 label serving as a proxy of xylem-transported CO2 entering the leaf from the branch. The uptake rate of the (13)C was manipulated by altering the vapor pressure deficit (VPD) (0.84, 1.29 and 1.83 kPa). Highest tissue enrichments were observed under the highest VPD. Among tissues, highest enrichment was observed in the petiole and the veins, regardless of the VPD treatment. Analysis of non-labeled leaves showed that some (13)C diffused from the labeled leaves and was fixed in the mesophyll of the non-labeled leaves. However, (13)C leaf tissue enrichment analysis with elemental analysis coupled to isotope ratio mass spectrometry was limited in spatial resolution at the leaf tissue level. Therefore, (11)C-based CO2 labeling combined with positron autoradiography was used and showed a more detailed spatial distribution within a single tissue, in particular in secondary veins. Therefore, in addition to (13)C, (11) C-based autoradiography can be used to study the fate of xylem-transported CO2 at leaf level, allowing the acquisition of data at a yet unprecedented resolution.

  14. 13C labelling reveals different contributions of photoassimilates from infructescences for fruiting in two temperate forest tree species.

    PubMed

    Hoch, G; Keel, S G

    2006-09-01

    The pathways of currently fixed carbon in fruit bearing branchlets were investigated in two temperate forest tree species (CARPINUS BETULUS and FAGUS SYLVATICA), which differ in texture of their vegetative infructescence tissues (leaf-like in CARPINUS vs. woody in FAGUS). During late spring, (13)C pulse-labelling was conducted on girdled, defoliated, girdled plus defoliated and untreated fruiting branchlets of mature trees IN SITU, to assess changes in C relations in response to the introduced C source-sink imbalances. At harvest in early August, 75 - 100 % of the recovered (13)C label was bound to infructescences (either fruits or vegetative infructescence tissue), revealing them as the prime C sinks for current photoassimilates. Leaves on girdled branchlets were not stronger labelled than on ungirdled ones in both species, indicating no upregulation of the leaves' photosynthetic capacity in response to the prevention of phloemic transport, which was also supported by measurements of light saturated photosynthesis. In contrast, (13)C labels tended to be higher after complete defoliation in the vegetative infructescence tissues of CARPINUS, suggesting enhanced net photosynthesis of green infructescence parts as compensation for the loss of regular leaves. The total labelling-derived (13)C content of whole infructescences was very similar between foliated and defoliated CARPINUS branchlets. Cupulae of FAGUS, on the other hand, remained almost unlabelled on defoliated branchlets, indicating the photosynthetic inactivity of this woody infructescence tissue. Consequently, CARPINUS still produced relatively high fruit masses on girdled plus defoliated branchlets, while in FAGUS fruit development ceased almost completely at this most severe treatment. Our results highlight that green vegetative infructescence tissue assimilates substantial amounts of C and can partly substitute regular leaves as C sources for successful fruit development. PMID:16883486

  15. Simultaneous CT-13C and VT-15N chemical shift labelling: application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH.

    PubMed

    Uhrín, D; Bramham, J; Winder, S J; Barlow, P N

    2000-11-01

    Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) 13C and variable time (VT) 15N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J(CC) = 28.6 ms). The magnetisation originating from NH protons is initially stored in the 2HzNz state, then prior to the VT chemical shift labelling period is converted into 2HzNy coherence. The VT -15N mode eliminates the effect of 1J(N,CO) and 1,2J(N,CA) coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH2 groups, thus removing any potential overlap with the CH3 signals. The CT-13CH3,VT-15N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH3 and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH3 and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.

  16. Carbon Transfer from the Host to Tuber melanosporum Mycorrhizas and Ascocarps Followed Using a 13C Pulse-Labeling Technique

    PubMed Central

    Le Tacon, François; Zeller, Bernd; Plain, Caroline; Hossann, Christian; Bréchet, Claude; Robin, Christophe

    2013-01-01

    Truffles ascocarps need carbon to grow, but it is not known whether this carbon comes directly from the tree (heterotrophy) or from soil organic matter (saprotrophy). The objective of this work was to investigate the heterotrophic side of the ascocarp nutrition by assessing the allocation of carbon by the host to Tuber melanosporum mycorrhizas and ascocarps. In 2010, a single hazel tree selected for its high truffle (Tuber melanosporum) production and situated in the west part of the Vosges, France, was labeled with 13CO2. The transfer of 13C from the leaves to the fine roots and T. melanosporum mycorrhizas was very slow compared with the results found in the literature for herbaceous plants or other tree species. The fine roots primarily acted as a carbon conduit; they accumulated little 13C and transferred it slowly to the mycorrhizas. The mycorrhizas first formed a carbon sink and accumulated 13C prior to ascocarp development. Then, the mycorrhizas transferred 13C to the ascocarps to provide constitutive carbon (1.7 mg of 13C per day). The ascocarps accumulated host carbon until reaching complete maturity, 200 days after the first labeling and 150 days after the second labeling event. This role of the Tuber ascocarps as a carbon sink occurred several months after the end of carbon assimilation by the host and at low temperature. This finding suggests that carbon allocated to the ascocarps during winter was provided by reserve compounds stored in the wood and hydrolyzed during a period of frost. Almost all of the constitutive carbon allocated to the truffles (1% of the total carbon assimilated by the tree during the growing season) came from the host. PMID:23741356

  17. Vibrational studies of {sup 13}C- and {sup 34}S-labelled bis(ethylenedithio)tetrathiafulvalene (ET) donor molecule

    SciTech Connect

    Ferraro, J.R.; Kini, A.M.; Williams, J.M.; Stout, P.

    1994-06-01

    FT-IR and FT-Raman studies of {sup 13}C- and {sup 34}S-labelled bis(ethylenedithio)-tetrathiafulvalene (BEDT-TTF or ET) electron-donor molecules were made and the results presented herein. Assignments for fundamental vibrations in ET were verified. Spectral data confirms that ET has no center-of-symmetry, and that the data can be reconciled by a D-type point group with only slight interactions occurring between the 4 molecules per unit cell.

  18. Survival of free-living Acholeplasma in aerated pig manure slurry revealed by 13C-labeled bacterial biomass probing

    PubMed Central

    Hanajima, Dai; Aoyagi, Tomo; Hori, Tomoyuki

    2015-01-01

    Many studies have been performed on microbial community succession and/or predominant taxa during the composting process; however, the ecophysiological roles of microorganisms are not well understood because microbial community structures are highly diverse and dynamic. Bacteria are the most important contributors to the organic-waste decomposition process, while decayed bacterial cells can serve as readily digested substrates for other microbial populations. In this study, we investigated the active bacterial species responsible for the assimilation of dead bacterial cells and their components in aerated pig manure slurry by using 13C-labeled bacterial biomass probing. After 3 days of forced aeration, 13C-labeled and unlabeled dead Escherichia coli cell suspensions were added to the slurry. The suspensions contained 13C-labeled and unlabeled bacterial cell components, possibly including the cell wall and membrane, as well as intracellular materials. RNA extracted from each slurry sample 2 h after addition of E. coli suspension was density-resolved by isopycnic centrifugation and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of bacterial 16S rRNA genes. In the heavy isotopically labeled RNA fraction, the predominant 13C-assimilating population was identified as belonging to the genus Acholeplasma, which was not detected in control heavy RNA. Acholeplasma spp. have limited biosynthetic capabilities and possess a wide variety of transporters, resulting in their metabolic dependence on external carbon and energy sources. The prevalence of Acholeplasma spp. was further confirmed in aerated pig manure slurry from four different pig farms by pyrosequencing of 16S rRNA genes; their relative abundance was ∼4.4%. Free-living Acholeplasma spp. had a competitive advantage for utilizing dead bacterial cells and their components more rapidly relative to other microbial populations, thus allowing the survival and prevalence

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

    PubMed

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

    2014-01-16

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

  20. 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

  1. Regioselective Syntheses of [13C]4-Labelled Sodium 1-Carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate and Sodium 2-Carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate from [13C]4-Maleic Anhydride

    PubMed Central

    Barsamian, Adam L.; Perkins, Matt J.; Field, Jennifer A.; Blakemore, Paul R.

    2014-01-01

    The entitled monohydrolysis products, also known as α- and β-ethylhexyl sulfosuccinate ('EHSS'), of the surfactant diisooctyl sulfosuccinate ('DOSS') were synthesized in stable isotope labelled form from [13C]4-maleic anhydride. Sodium [13C]4-1-carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate (α-EHSS) was prepared by the method of Larpent by reaction of 2-ethylhexan-1-ol with [13C]4-maleic anhydride followed by regioselective conjugate addition of sodium bisulfite to the resulting monoester (38% overall yield). The regiochemical outcome of bisulfite addition was confirmed by a combination of 13C/13C (INADEQUATE) and 1H/13C (HMBC) NMR spectral correlation experiments. Sodium [13C]4-2-carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate (β-EHSS) was prepared in four steps by reaction of 4-methoxybenzyl alcohol (PMBOH) with [13C]4-maleic anhydride, regioselective sodium bisulfite addition, DCC mediated esterification with 2-ethylhexan-1-ol, and PMB ester deprotection with trifluoroacetic acid (13% overall yield). The regiochemical outcome of the second synthesis was confirmed by a combination of 1JCC scalar coupling constant analysis and 1H/13C (HMBC) NMR spectral correlation. The materials prepared are required as internal standards for the LC-MS/MS trace analysis of the degradation products of DOSS, the anionic surfactant found in Corexit, the oil dispersant used during emergency response efforts connected to the Deepwater Horizon oil spill of April 2010. PMID:24700711

  2. Nitrogen and Carbon Flows Estimated by 15N and 13C Pulse-Chase Labeling during Regrowth of Alfalfa.

    PubMed Central

    Avice, J. C.; Ourry, A.; Lemaire, G.; Boucaud, J.

    1996-01-01

    The flow of 15N and 13C from storage compounds in organs remaining after defoliation (sources) to regrowing tissue (sinks), and 13C losses through root or shoot respiration were assessed by pulse-chase labeling during regrowth of alfalfa (Medicago sativa L.) following shoot removal. A total of 73% of labeled C and 34% of labeled N were mobilized in source organs within 30 d. Although all of the 15N from source organs was recovered in the regrowing tissue, much of the 13C was lost, mainly as CO2 respired from the root (61%) or shoot (8%), and was found to a lesser extent in sink tissue (5%). After 3, 10, or 30 d of regrowth, 87, 66, and 52% of shoot N, respectively, was derived from source tissue storage compounds; the rest resulted from translocation of fixed N2. Overall results suggest that most shoot C was linked to photosynthetic activity rather than being derived from mobilization of stored C in source organs. Furthermore, isotopic analysis of different chemical fractions of plant tissue suggests that between 14 and 58% of the shoot C derived from source tissues was linked to the mobilization of N compounds, not carbohydrates. PMID:12226391

  3. Vitamin K absorption and kinetics in human subjects after consumption of 13C-labelled phylloquinone from kale.

    PubMed

    Novotny, Janet A; Kurilich, Anne C; Britz, Steven J; Baer, David J; Clevidence, Beverly A

    2010-09-01

    The absorption and plasma disappearance of vitamin K were investigated by uniformly labelling phylloquinone in kale with carbon-13, and by feeding the kale to study subjects. Seven healthy volunteers ingested a single 400 g serving of kale with 30 g vegetable oil. The kale provided 156 nmol of phylloquinone. Serial plasma samples were collected and analysed for the appearance of 13C-phylloquinone by HPLC-MS. Six of the subjects showed significant amounts of labelled phylloquinone in plasma, though one subject's plasma was not consistently enriched above the detection limit, and this subject's baseline plasma phylloquinone level was the lowest in the group. After ingestion of the labelled kale, plasma 13C-phylloquinone concentration increased rapidly to a peak between 6 and 10 h, and then rapidly decreased. Average peak plasma concentration for the six subjects with detectable 13C-phylloquinone was 2.1 nmol/l. Plasma concentration-time data were analysed by compartmental modelling. Modelling results demonstrated a mean (n 6) bioavailability of phylloquinone from kale to be 4.7%. Plasma and tissue half-times for phylloquinone were found to be 8.8 and 215 h, respectively.

  4. 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

  5. Optimization of 13C dynamic nuclear polarization: isotopic labeling of free radicals

    NASA Astrophysics Data System (ADS)

    Niedbalski, Peter; Parish, Christopher; Kiswandi, Andhika; Lumata, Lloyd

    Dynamic nuclear polarization (DNP) is a physics technique that amplifies the nuclear magnetic resonance (NMR) signals by transferring the high polarization of the electrons to the nuclear spins. Thus, the choice of free radical is crucial in DNP as it can directly affect the NMR signal enhancement levels, typically on the order of several thousand-fold in the liquid-state. In this study, we have investigated the efficiency of four variants of the well-known 4-oxo-TEMPO radical (normal 4-oxo-TEMPO plus its 15N-enriched and/or perdeuterated variants) for use in DNP of an important metabolic tracer [1-13C]acetate. Though the variants have significant differences in electron paramagnetic resonance (EPR) spectra, we have found that changing the composition of the TEMPO radical through deuteration or 15N doping yields no significant difference in 13C DNP efficiency at 3.35 T and 1.2 K. On the other hand, deuteration of the solvent causes a significant increase of 13C polarization that is consistent over all the 4-oxo-TEMPO variants. These findings are consistent with the thermal mixing model of DNP. This work is supported by US Dept of Defense Award No. W81XWH-14-1-0048 and the Robert A. Welch Foundation Grant No. AT-1877.

  6. 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

  7. Site-specific φ- and ψ-torsion angle determination in a uniformly/extensively 13C- and 15N-labeled peptide

    NASA Astrophysics Data System (ADS)

    Wi, Sungsool; Spano, Justin

    2011-10-01

    A solid-state rotational-echo double resonance (REDOR) NMR method was introduced to identify the ϕ- and ψ-torsion angle from a 1H- 15N or 1H- 13C' spin system of alanine-like residues in a selectively, uniformly, or extensively 15N-/ 13C-labeled peptide. When a C α( i) or a 15N peak is site-specifically obtainable in the NMR spectrum of a uniformly 15N/ 13C-labeled sample system, the ψ- or ϕ-torsion angle specified by the conformational structure of peptide geometry involving 15N( i)- 1H αi - 15N( i + 1) or 13C'( i - 1)- 1H Ni- 13C'( i) spin system can be identified based on 13C α- or 15N-detected 1H α- 15N or 1H N- 13C REDOR experiment. This method will conveniently be utilized to identify major secondary motifs, such as α-helix, β-sheet, and β-turn, from a uniformly 15N-/ 13C-labled peptide sample system. When tested on a 13C-/ 15N-labeled model system of a three amino acid peptide Gly-[U- 13C, 15N]Ala-[U- 13C, 15N]Leu, the ψ-angle of alanine obtained experimentally, ψ = -40 ± 30°, agreed reasonably well with the X-ray determined angle, ψ = -39°.

  8. 13C labelled cholesteryl octanoate breath test for assessing pancreatic exocrine insufficiency

    PubMed Central

    Ventrucci, M; Cipolla, A; Ubalducci, G; Roda, A; Roda, E

    1998-01-01

    Background—A non-invasive test for assessment of fat digestion has been developed based on the intraluminal hydrolysis of cholesteryl-[1-13C]octanoate by pancreatic esterase. 
Aims—To determine the diagnostic performance of this breath test in the assessment of exocrine pancreatic function. 
Methods—The test was performed in 20 healthy controls, 22 patients with chronic pancreatic disease (CPD), four with biliopancreatic diversion (BPD), and 32 with non-pancreatic digestive diseases (NPD); results were compared with those of other tubeless tests (faecal chymotrypsin and fluorescein dilaurate test). 
Results—Hourly recoveries of 13CO2 were significantly lower in CPD when compared with healthy controls or NPD. In patients with CPD with mild to moderate insufficiency, the curve of 13CO2 recovery was similar to that of healthy controls, while in those with severe insufficiency it was flat. In three patients with CPD with severe steatorrhoea, a repeat test after pancreatic enzyme supplementation showed a significant rise in 13CO2 recovery. The four BPD patients had low and delayed 13CO2 recovery. Only eight of the 32 patients with NPD had abnormal breath test results. There was a significant correlation between the results of the breath test and those of faecal chymotrypsin, the fluorescein dilaurate test, and faecal fat measurements. For the diagnosis of pancreatic disease using the three hour cumulative 13CO2 recovery test, the sensitivity was 68.2% and specificity 75.0%; values were similar to those of the other two tubeless pancreatic function tests. In seven healthy controls, nine patients with CPD, and nine with NPD a second breath test was performed using Na-[1-13C]octanoate and a pancreatic function index was calculated as the ratio of 13C recovery obtained in the two tests: at three hours this index was abnormal in eight patients with CPD and in three with NPD. 
Conclusion—The cholesteryl-[1-13C]octanoate breath test can be useful for the

  9. Selective {sup 2}H and {sup 13}C labeling in NMR analysis of solution protein structure and dynamics

    SciTech Connect

    LeMaster, D.M.

    1994-12-01

    Preparation of samples bearing combined isotope enrichment patterns has played a central role in the recent advances in NMR analysis of proteins in solution. In particular, uniform {sup 13}C, {sup 15}N enrichment has made it possible to apply heteronuclear multidimensional correlation experiments for the mainchain assignments of proteins larger than 30 KDa. In contrast, selective labeling approaches can offer advantages in terms of the directedness of the information provided, such as chirality and residue type assignments, as well as through enhancements in resolution and sensitivity that result from editing the spectral complexity, the relaxation pathways and the scalar coupling networks. In addition, the combination of selective {sup 13}C and {sup 2}H enrichment can greatly facilitate the determination of heteronuclear relaxation behavior.

  10. Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino

  11. Atmospheric CO2 level affects plants' carbon use efficiency: insights from a 13C labeling experiment on sunflower stands

    NASA Astrophysics Data System (ADS)

    Gong, Xiaoying; Schäufele, Rudi; Schnyder, Hans

    2015-04-01

    The increase of atmospheric CO2 concentration has been shown to stimulate plant photosynthesis and (to a lesser extent) growth, thereby acting as a possible sink for the additional atmospheric CO2. However, this effect is dependent on the efficiency with which plants convert atmospheric carbon into biomass carbon, since a considerable proportion of assimilated carbon is returned to the atmosphere via plant respiration. As a core parameter for carbon cycling, carbon use efficiency of plants (CUE, the ratio of net primary production to gross primary production) quantifies the proportion of assimilated carbon that is incorporated into plant biomass. CUE has rarely been assessed based on measurements of complete carbon balance, due to methodological difficulties in measuring respiration rate of plants in light. Moreover, foliar respiration is known to be inhibited in light, thus foliar respiration rate is generally lower in light than in dark. However, this phenomenon, termed as inhibition of respiration in light (IRL), has rarely been assessed at the stand-scale and been incorporated into the calculation of CUE. Therefore, how CUE responses to atmospheric CO2 levels is still not clear. We studied CUE of sunflower stands grown at sub-ambient CO2 level (200 μmol mol-1) and elevated CO2 level (1000 μmol mol-1) using mesocosm-scale gas exchange facilities which enabled continuous measurements of 13CO2/12CO2 exchange. Appling steady-state 13C labeling, fluxes of respiration and photosynthesis in light were separated, and tracer kinetic in respiration was analyzed. This study provides the first data on CUE at a mesocosm-level including respiration in light in different CO2 environments. We found that CUE of sunflower was lower at an elevated CO2 level than at a sub-ambient CO2 level; and the ignorance of IRL lead to erroneous estimations of CUE. Variation in CUE at atmospheric CO2 levels was attributed to several mechanisms. In this study, CO2 enrichment i) affected the

  12. Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine

    NASA Astrophysics Data System (ADS)

    Pettelkau, Jens; Ihling, Christian H.; Frohberg, Petra; van Werven, Lars; Jahn, Olaf; Sinz, Andrea

    2014-09-01

    We describe the use of the 13C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein-peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a 13C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein-protein interaction studies.

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

    NASA Astrophysics Data System (ADS)

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

    2003-12-01

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

  14. Biosynthesis of pyrroloquinoline quinone. 1. Identification of biosynthetic precursors using /sup 13/C labeling and NMR spectroscopy

    SciTech Connect

    Houck, D.R.; Hanners, J.L.; Unkefer, C.J.

    1988-09-28

    The biosynthesis of pyrroloquinoline quinone (PQQ) in the methylotropic bacterium methylobacterium AM1 has been investigated using /sup 13/C-labelling of the products and NMR spectroscopy. The data indicated that the quinoline portion of PQQ is formed by a novel condensation of N-1, C-2, -3, and -4 of glutamate with a symmetrical six-carbon ring derived from the shikimate pathway. It is postulated that tyrosine is the shikimate-derived percursor, since pyrrole could be formed by the internal cyclization of the amino acid backbone. 18 references, 2 figures, 2 tables.

  15. Fast pyrolysis of 13C-labeled cellobioses: gaining insights into the mechanisms of fast pyrolysis of carbohydrates.

    PubMed

    Degenstein, John C; Murria, Priya; Easton, Mckay; Sheng, Huaming; Hurt, Matt; Dow, Alex R; Gao, Jinshan; Nash, John J; Agrawal, Rakesh; Delgass, W Nicholas; Ribeiro, Fabio H; Kenttämaa, Hilkka I

    2015-02-01

    A fast-pyrolysis probe/tandem mass spectrometer combination was utilized to determine the initial fast-pyrolysis products for four different selectively (13)C-labeled cellobiose molecules. Several products are shown to result entirely from fragmentation of the reducing end of cellobiose, leaving the nonreducing end intact in these products. These findings are in disagreement with mechanisms proposed previously. Quantum chemical calculations were used to identify feasible low-energy pathways for several products. These results provide insights into the mechanisms of fast pyrolysis of cellulose.

  16. Belowground carbon allocation in a temperate beech forest: new insight into carbon residence time using whole tree 13C labelling

    NASA Astrophysics Data System (ADS)

    Epron, D.; Ngao, J.; Plain, C.; Longdoz, B.; Granier, A.

    2011-12-01

    Belowground carbon allocation is an important component of forest carbon budget, affecting tree growth (competition between aboveground and belowground carbon sinks), acquisition of belowground resources (nutrients and water) that are often limiting forest ecosystems and soil carbon sequestration. Total belowground carbon flow can be estimated using a mass-balance approach as cumulative soil CO2 efflux minus the carbon input from aboveground litter plus the changes in the C stored in roots, in the forest floor, and in the soil, and further compared to gross annual production. While this approach is useful for understanding the whole ecosystem carbon budget, uncertainties remain about the contribution of the different belowground pools of carbon to ecosystem respiration and carbon sequestration. New insights into transfer rate and residence time of carbon in belowground compartments can be gained from in situ whole-crown 13C labelling experiments. We combined both approaches in a young temperate beech forest in north-eastern France where ecosystem carbon fluxes are recorded since a decade. Carbon allocated belowground represented less than 40% of gross primary production in this young beech forest. Autotrophic respiration assessed by comparing soil CO2 efflux measured on normal and on root exclusion plots, accounted for 60% of the total belowground carbon flow. This indicated a rather short mean residence time of carbon allocated belowground in the soil compartments. The recovery of 13C in soil CO2 efflux after pulse-labelling entire crowns of tree with 13CO2 at several occasions during the growing season was observed a few couple of hours after the labelling. That indicates a rapid transfer of 13C belowground with a maximum occurring within 2 to 4 days after labelling. Label was recovered at the same time in the respiration and in the biomass of both fine roots and soil microbes. Allocation of recently assimilated carbon to soil microbial respiration was greater in

  17. Validating the Incorporation of 13C and 15N in a Shorebird That Consumes an Isotopically Distinct Chemosymbiotic Bivalve

    PubMed Central

    van Gils, Jan A.; Ahmedou Salem, Mohamed Vall

    2015-01-01

    The wealth of field studies using stable isotopes to make inferences about animal diets require controlled validation experiments to make proper interpretations. Despite several pleas in the literature for such experiments, validation studies are still lagging behind, notably in consumers dwelling in chemosynthesis-based ecosystems. In this paper we present such a validation experiment for the incorporation of 13C and 15N in the blood plasma of a medium-sized shorebird, the red knot (Calidris canutus canutus), consuming a chemosymbiotic lucinid bivalve (Loripes lucinalis). Because this bivalve forms a symbiosis with chemoautotrophic sulphide-oxidizing bacteria living inside its gill, the bivalve is isotopically distinct from ‘normal’ bivalves whose food has a photosynthetic basis. Here we experimentally tested the hypothesis that isotope discrimination and incorporation dynamics are different when consuming such chemosynthesis-based prey. The experiment showed that neither the isotopic discrimination factor, nor isotopic turnover time, differed between birds consuming the chemosymbiotic lucinid and a control group consuming a photosynthesis-based bivalve. This was true for 13C as well as for 15N. However, in both groups the 15N discrimination factor was much higher than expected, which probably had to do with the birds losing body mass over the course of the experiment. PMID:26458005

  18. Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry.

    PubMed

    Heinzle, Elmar; Yuan, Yongbo; Kumar, Sathish; Wittmann, Christoph; Gehre, Matthias; Richnow, Hans-Herrmann; Wehrung, Patrick; Adam, Pierre; Albrecht, Pierre

    2008-09-15

    The applicability of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of 13C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the 13C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-13C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the 13C enrichment. The approach to correct for these effects in metabolic flux studies using delta13C measurement by GC-C-IRMS uses two parallel experiments applying substrate with natural abundance and 13C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of 13C in proteinogenic amino acids over the whole range of applied concentrations of [1-13C]glucose. The current findings indicate a great potential of GC-C-IRMS for labeling quantification in 13C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.

  19. Molecular Investigation of the Short-term Sequestration of Natural Abundance 13C -labelled Cow Dung in the Surface Horizons of a Temperate Grassland Soil

    NASA Astrophysics Data System (ADS)

    Dungait, J.; Bol, R.; Evershed, R. P.

    2004-12-01

    An adequate understanding of the carbon (C) sequestration potential of grasslands requires that the quantity and residence times of C inputs be measured. Herbivore dung is largely comprised of plant cell wall material, a significant source of stable C in intensively grazed temperate grassland ecosystems that contributes to the soil carbon budget. Our work uses compound-specific isotope analysis to identify the pattern of input of dung-derived compounds from natural abundance 13C/-labelled cow dung into the surface horizons of a temperate grassland soil over one year. C4 dung (δ 13C \\-12.6 ‰ ) from maize fed cows was applied to a temperate grassland surface (δ 13C \\-29.95 ‰ ) at IGER-North Wyke (Devon, UK), and dung remains and soil cores beneath the treatments collected at ŧ = 7, 14, 28, 56, 112, 224 and 372 days. Bulk dung carbon present in the 0\\-1 cm and 1\\-5 cm surface horizons of a grassland soil over one year was estimated using Δ 13C between C4 dung and C3 dung, after Bol {\\et al.} (2000). The major biochemical components of dung were quantified using proximate forage fibre analyses, after Goering and Van Soest (1970) and identified using `wet' chemical and GC-MS methods. Plant cell wall polysaccharides and lignin were found to account for up to 67 {%} of dung dry matter. Hydrolysed polysaccharides were prepared as alditol acetates for analyses (after Docherty {\\et al.}, 2001), and a novel application of an off-line pyrolysis method applied to measure lignin-derived phenolic compounds (after Poole & van Bergen, 2002). This paper focuses on major events in the incorporation of dung carbon, estimated using natural abundance 13C&-slash;labelling technique. This revealed a major bulk input of dung carbon after a period of significant rainfall with a consequent decline in bulk soil δ 13C values until the end of the experiment (Dungait {\\et al.}, submitted). Findings will be presented revealing contribution of plant cell wall polysaccharides and

  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. Assimilation of xylem-transported 13C-labelled CO2 in leaves and branches of sycamore (Platanus occidentalis L.).

    PubMed

    McGuire, M A; Marshall, J D; Teskey, R O

    2009-01-01

    Previous reports have shown that CO(2) dissolved in xylem sap in tree stems can move upward in the transpiration stream. To determine the fate of this dissolved CO(2), the internal transport of respired CO(2) at high concentration from the bole of the tree was simulated by allowing detached young branches of sycamore (Platanus occidentalis L.) to transpire water enriched with a known quantity of (13)CO(2) in sunlight. Simultaneously, leaf net photosynthesis and CO(2) efflux from woody tissue were measured. Branch and leaf tissues were subsequently analysed for (13)C content to determine the quantity of transported (13)CO(2) label that was fixed. Treatment branches assimilated an average of 35% (SE=2.4) of the (13)CO(2) label taken up in the treatment water. The majority was fixed in the woody tissue of the branches, with smaller amounts fixed in the leaves and petioles. Overall, the fixation of internally transported (13)CO(2) label by woody tissues averaged 6% of the assimilation of CO(2) from the atmosphere by the leaves. Woody tissue assimilation rates calculated from measurements of (13)C differed from rates calculated from measurements of CO(2) efflux in the lower branch but not in the upper branch. The results of this study showed unequivocally that CO(2) transported in xylem sap can be fixed in photosynthetic cells in the leaves and branches of sycamore trees and provided evidence that recycling of xylem-transported CO(2) may be an important means by which trees reduce the carbon cost of respiration.

  2. 13C-labeled oligosaccharides in breastfed infants' urine: individual-, structure- and time-dependent differences in the excretion.

    PubMed

    Dotz, Viktoria; Rudloff, Silvia; Blank, Dennis; Lochnit, Günter; Geyer, Rudolf; Kunz, Clemens

    2014-02-01

    Human milk oligosaccharides (HMOs) have been paid much attention due to their beneficial effects observed in vitro, e.g., prebiotic, anti-infective and anti-inflammatory properties. However, in vivo investigations with regard to HMO metabolism and functions are rare. The few data available indicate that HMOs are absorbed to a low extent and excreted via urine without noteworthy modifications, whereas the major proportion reaches infant's colon undigested. Via intrinsic (13)C-labeling of HMOs during their biosynthesis in the mammary gland of 10 lactating women, we were able to follow the fate of (13)C-labeled oligosaccharides (OSs) from their secretion in milk to the excretion in the urine of their breastfed infants. To a certain extent, we could therefore discriminate between original HMOs and non-labeled OSs derived from degradation of HMOs or endogenous glycoconjugates. By means of our novel, rapid, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based approach, we found a homogeneous time pattern of isotopomer enrichment in milk among all subjects and between single OS species. In contrast, the time curves from infants' urine varied strongly between individuals and OS species, though the overall MALDI-TOF MS profile resembled those of the mothers' milk. Our data suggest that neutral HMOs might be processed and/or utilized differentially after or upon absorption from the gut, as deduced from their structure-dependent variation in the extent of tracer enrichment and in the retention times in infant's organism. This sheds new light on the role of HMOs within infant's body, beyond the intestine and its microbiota alone.

  3. Investigation of the degradation of 13C-labeled fungal biomass in soil - fate of carbon in a soil bioreactor system

    NASA Astrophysics Data System (ADS)

    Schweigert, Michael; Fester, Thomas; Miltner, Anja; Kästner, Matthias

    2014-05-01

    Nutrient balances and degradation processes in boreal forests are mainly influenced by interactions of plant roots and ectomycorrhizal fungi. Plants benefit from nitrogen compounds provided by their symbiotic interaction partner. In return ectomycorrhiza are provided by large amounts of carbon from the plants which is used for the synthesis of hyphal networks in soil and for metabolic activity for nutrient uptake. Therefore ectomycorrhizal fungi play a major role in ecosystems of boreal forests and are consequently an important sink for carbon by building large amounts of mycelia. Recently, it has been shown that microbial biomass residues contribute significantly to soil organic matter formation. This suggests that also residues of ectomycorrhizal fungi may be an important source for soil organic matter formation in forest soils where these fungi are abundant. However, the fate of ectomycorrhizal biomass residues in soils is unknown. We therefore investigated the fate of ectomycorrhizal biomass in soil in a bioreactor system to quantify the contribution of this material to soil organic matter formation. As a model organism, we selected Laccaria bicolor, which was labelled by growing the fungus on 13C glucose. The stable isotope-labeled biomass was then homogenized and incubated in a podzol from a typical forest site in Central Germany. The fate of the labeled biomass was traced by analyzing the amount of 13C mineralized and the amount remaining in the soil. The fungal biomass carbon was mineralized rather rapidly during the first 25 days. Then the mineralization rate slowed down, but mineralization continued until the end of the experiment, when approximately 40% of the 13C was mineralized and 60% remained in soil. In addition, we analyzed biomolecules such as fatty acids to trace the incorporation of the L. bicolor-derived biomass carbon into other microorganisms and to identify potential primary consumers of fungal biomass. By these analyses, we found a

  4. Investigation of the degradation of 13C-labeled fungal biomass in soil - fate of carbon in a soil bioreactor system

    NASA Astrophysics Data System (ADS)

    Schweigert, Michael; Fester, Thomas; Miltner, Anja; Kaestner, Matthias

    2015-04-01

    Nutrient balances and degradation processes in boreal forests are mainly influenced by interactions of plant roots and ectomycorrhizal fungi. Plants benefit from nitrogen compounds provided by their symbiotic interaction partner. In return ectomycorrhiza are provided by large amounts of carbon from the plants which is used for the synthesis of hyphal networks in soil and for metabolic activity for nutrient uptake. Therefore, ectomycorrhizal fungi play a major role in ecosystems of boreal forests and are consequently an important sink for carbon by building large amount of mycelia. Recently, it has been shown that microbial biomass residues contribute significantly to soil organic matter formation. This suggests that also residues of ectomycorrhizal fungi may be an important source for soil organic matter formation in forest soils where these fungi are abundant. However, the fate of ectomycorrhizal biomass residues in soils is unknown. We therefore investigated the fate of ectomycorrhizal biomass in soil in a soil bioreactor system to quantify the contribution of this material to soil organic matter formation. As a model organism, we selected Laccaria bicolor, which was labelled by growing the fungus on 13C glucose. The stable isotope-labeled biomass was then homogenized and incubated in a podzol from a typical forest site in Central Germany. The fate of the labeled biomass was traced by analyzing the amount of 13C mineralized and the amount remaining in the soil. The fungal biomass carbon was mineralized rather rapidly during the first 50 days. Then the mineralization rate slowed down, but mineralization continued until the end of the experiment, when approximately 40% of the 13C was mineralized and 60% remained in soil. In addition, we analyzed biomolecules such as fatty acids to trace the incorporation of the L. bicolor-derived biomass carbon into other microorganisms and to identify potential primary consumers of fungal biomass. By these analyses, we found a

  5. A solution NMR study of the selectively 13C, 15N-labeled peptaibol chrysospermin C in methanol.

    PubMed

    Anders, R; Wenschuh, H; Soskic, V; Fischer-Frühholz, S; Ohlenschläger, O; Dornberger, K; Brown, L R

    1998-07-01

    The conformation of the 19-residue peptaibol chrysospermin C in methanol has been investigated by NMR spectroscopy using selective 15N and 13C labeling of the alpha-aminoisobutyric acid (Aib) residues. Complete 1H and 13C sequential assignments, including stereospecific assignments for the heavily overlapped resonances from the two Cbeta methyl groups of the eight Aib residues, are reported for a peptaibol for the first time. An Aib residue followed by a Pro is an exception to previous suggestions regarding stereospecific assignment of the two Cbeta methyl groups of Aib residues. Local nuclear Overhauser effects and 3J(HNC') and 3J(HNCbeta) scalar couplings indicate that the phi angles of the Aib residues are restricted sterically to local conformations consistent with right-handed helices. Despite these constraints on the eight Aib residues, the NMR data for chrysospermin C in methanol are generally most consistent with an ensemble of transient conformations, including backbone conformations inconsistent with helical structures. Initial NMR measurements for chrysospermin C bound to micelles suggest structural and dynamic differences relative to alamethicin bound to micelles which may be related to differences in gating voltages for formation of ion channels.

  6. 3D HCCH 3-TOCSY for Resonance Assignment of Methyl-Containing Side Chains in 13C-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Uhrín, Dušan; Uhrínová, Stanislava; Leadbeater, Claire; Nairn, Jacqueline; Price, Nicholas C.; Barlow, Paul N.

    2000-02-01

    Two 3D experiments, (H)CCH3-TOCSY and H(C)CH3-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t1), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t2) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t3). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t2max = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH2, or CH2 and CH, resonances from the F2F3 planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a 15N, 13C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).

  7. 13C-TRIPLY Labeled Ethyl Cyanide Submillimeterwave Study with Lille's Fast Scan Dds-Based Spectrometer

    NASA Astrophysics Data System (ADS)

    Pienkina, A.; Motiyenko, R. A.; Margulès, L.; Müller, Holger S. P.; Guillemin, J.-C.

    2016-06-01

    This study of the 13C-triply labeled species of ethyl cyanide (CH_3CH_2CN) follows our recent work on the three 13C-doubly-labeled that allowed their detection in the line survey recently obtained with ALMA (EMoCA). The detection of isotopologues could improve the knowledge of the astrochemistry. The other goal is to clean the surveys from the lines of known molecules in order to detect new ones, this is especially important for the abundant complex organic molecules like ethyl cyanide. As in the case of the doubly substitued species, no spectroscopic studies exist up to now for 13CH_313CH_213CN, the first predictions were thus obtained from scaled ab initio calculations. The spectra were recorded and analyzed up to 1 THz. More than 5500 lines were fitted with quantum numbers J and K_a up to 95 and 25 respectively. The spectra were obtained with the new version of the Lille's solid state spectrometers. This new version used Direct Digital Synthesizer in order to speed up acquisition time. We constructed a spectrometer covering a decade, from 150 to 1500 GHz, it scans the full range in 24 hours with high sensitivity and accuracy. This work was supported by the CNES and the Action sur Projets de l'INSU, PCMI. This work was also done under ANR-13-BS05-0008-02 IMOLABS Margules, L.; et al. 2015, 69th International Symposium on Molecular Spectroscopy, RI06 Belloche, A.; et al. 2014, Science, 345, 1584

  8. Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma*

    PubMed Central

    Priego-Capote, Feliciano; Scherl, Alexander; Müller, Markus; Waridel, Patrice; Lisacek, Frédérique; Sanchez, Jean-Charles

    2010-01-01

    Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [13C6]glucose incubation to evaluate the native glycated proteins labeled with [12C6]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive

  9. Synthesis of aromatic (13)C/(2)H-α-ketoacid precursors to be used in selective phenylalanine and tyrosine protein labelling.

    PubMed

    Lichtenecker, R J

    2014-10-14

    Recent progress in protein NMR spectroscopy revealed aromatic residues to be valuable information sources for performing structure and motion analysis of high molecular weight proteins. However, the applied NMR experiments require tailored isotope labelling patterns in order to regulate spin-relaxation pathways and optimize magnetization transfer. We introduced a methodology to use α-ketoacids as metabolic amino acid precursors in cell-based overexpression of phenylalanine and/or tyrosine labelled proteins in a recent publication, which we have now developed further by providing synthetic routes to access the corresponding side-chain labelled precursors. The target compounds allow for selective introduction of (13)C-(1)H spin systems in a highly deuterated chemical environment and feature alternating (12)C-(13)C-(12)C ring-patterns. The resulting isotope distribution is especially suited to render straightforward (13)C spin relaxation experiments possible, which provide insight into the dynamic properties of the corresponding labelled proteins.

  10. 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.

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

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

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

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

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin. PMID:21818779

  14. Identification of aquatically available carbon from algae through solution-state NMR of whole (13)C-labelled cells.

    PubMed

    Akhter, Mohammad; Dutta Majumdar, Rudraksha; Fortier-McGill, Blythe; Soong, Ronald; Liaghati-Mobarhan, Yalda; Simpson, Myrna; Arhonditsis, George; Schmidt, Sebastian; Heumann, Hermann; Simpson, André J

    2016-06-01

    Green algae and cyanobacteria are primary producers with profound impact on food web functioning. Both represent key carbon sources and sinks in the aquatic environment, helping modulate the dissolved organic matter balance and representing a potential biofuel source. Underlying the impact of algae and cyanobacteria on an ecosystem level is their molecular composition. Herein, intact (13)C-labelled whole cell suspensions of Chlamydomonas reinhardtii, Chlorella vulgaris and Synechocystis were studied using a variety of 1D and 2D (1)H/(13)C solution-state nuclear magnetic resonance (NMR) spectroscopic experiments. Solution-state NMR spectroscopy of whole cell suspensions is particularly relevant as it identifies species that are mobile (dissolved or dynamic gels), 'aquatically available' and directly contribute to the aquatic carbon pool upon lysis, death or become a readily available food source on consumption. In this study, a wide range of metabolites and structural components were identified within the whole cell suspensions. In addition, significant differences in the lipid/triacylglyceride (TAG) content of green algae and cyanobacteria were confirmed. Mobile species in algae are quite different from those in abundance in 'classic' dissolved organic matter (DOM) indicating that if algae are major contributors to DOM, considerable selective preservation of minor components (e.g. sterols) or biotransformation would have to occur. Identifying the metabolites and dissolved components within algal cells by NMR permits future studies of carbon transfer between species and through the food chain, whilst providing a foundation to better understand the role of algae in the formation of DOM and the sequestration/transformation of carbon in aquatic environments. PMID:27074782

  15. A 13C labelling study on carbon fluxes in Arctic plankton communities under elevated CO2 levels

    NASA Astrophysics Data System (ADS)

    de Kluijver, A.; Soetaert, K.; Czerny, J.; Schulz, K. G.; Boxhammer, T.; Riebesell, U.; Middelburg, J. J.

    2013-03-01

    The effect of CO2 on carbon fluxes (production, consumption, and export) in Arctic plankton communities was investigated during the 2010 EPOCA (European project on Ocean Acidification) mesocosm study off Ny Ålesund, Svalbard. 13C labelled bicarbonate was added to nine mesocosms with a range in pCO2 (185 to 1420 μatm) to follow the transfer of carbon from dissolved inorganic carbon (DIC) into phytoplankton, bacterial and zooplankton consumers, and export. A nutrient-phytoplankton-zooplankton-detritus model amended with 13C dynamics was constructed and fitted to the data to quantify uptake rates and carbon fluxes in the plankton community. The plankton community structure was characteristic for a post-bloom situation and retention food web and showed high bacterial production (∼31% of primary production), high abundance of mixotrophic phytoplankton, low mesozooplankton grazing (∼6% of primary production) and low export (∼7% of primary production). Zooplankton grazing and export of detritus were sensitive to CO2: grazing decreased and export increased with increasing pCO2. Nutrient addition halfway through the experiment increased the export, but not the production rates. Although mixotrophs showed initially higher production rates with increasing CO2, the overall production of POC (particulate organic carbon) after nutrient addition decreased with increasing CO2. Interestingly, and contrary to the low nutrient situation, much more material settled down in the sediment traps at low CO2. The observed CO2 related effects potentially alter future organic carbon flows and export, with possible consequences for the efficiency of the biological pump.

  16. Identification of aquatically available carbon from algae through solution-state NMR of whole (13)C-labelled cells.

    PubMed

    Akhter, Mohammad; Dutta Majumdar, Rudraksha; Fortier-McGill, Blythe; Soong, Ronald; Liaghati-Mobarhan, Yalda; Simpson, Myrna; Arhonditsis, George; Schmidt, Sebastian; Heumann, Hermann; Simpson, André J

    2016-06-01

    Green algae and cyanobacteria are primary producers with profound impact on food web functioning. Both represent key carbon sources and sinks in the aquatic environment, helping modulate the dissolved organic matter balance and representing a potential biofuel source. Underlying the impact of algae and cyanobacteria on an ecosystem level is their molecular composition. Herein, intact (13)C-labelled whole cell suspensions of Chlamydomonas reinhardtii, Chlorella vulgaris and Synechocystis were studied using a variety of 1D and 2D (1)H/(13)C solution-state nuclear magnetic resonance (NMR) spectroscopic experiments. Solution-state NMR spectroscopy of whole cell suspensions is particularly relevant as it identifies species that are mobile (dissolved or dynamic gels), 'aquatically available' and directly contribute to the aquatic carbon pool upon lysis, death or become a readily available food source on consumption. In this study, a wide range of metabolites and structural components were identified within the whole cell suspensions. In addition, significant differences in the lipid/triacylglyceride (TAG) content of green algae and cyanobacteria were confirmed. Mobile species in algae are quite different from those in abundance in 'classic' dissolved organic matter (DOM) indicating that if algae are major contributors to DOM, considerable selective preservation of minor components (e.g. sterols) or biotransformation would have to occur. Identifying the metabolites and dissolved components within algal cells by NMR permits future studies of carbon transfer between species and through the food chain, whilst providing a foundation to better understand the role of algae in the formation of DOM and the sequestration/transformation of carbon in aquatic environments.

  17. Sequential backbone assignment of uniformly 13C-labeled RNAs by a two-dimensional P(CC)H-TOCSY triple resonance NMR experiment.

    PubMed

    Wijmenga, S S; Heus, H A; Leeuw, H A; Hoppe, H; van der Graaf, M; Hilbers, C W

    1995-01-01

    A new 1H-13C-31P triple resonance experiment is described which allows unambiguous sequential backbone assignment in 13C-labeled oligonucleotides via through-bond coherence transfer from 31P via 13C to 1H. The approach employs INEPT to transfer coherence from 31P to 13C and homonuclear TOCSY to transfer the 13C coherence through the ribose ring, followed by 13C to 1H J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route involves transfer of 31Pi coherence via C4'i and C4'i-1, because of the relatively large JPC4' couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4'i and C4'i-1 coherences are subsequently transferred to, amongst others, H1'i and H1'i-1, respectively, leading to a 2D 1H-31P spectrum which allows a sequential assignment in the 31P-1H1' region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on a 13C-labeled RNA hairpin with the sequence 5'(GGGC-CAAA-GCCU)3'.

  18. A method for (13)C-labeling of metabolic carbohydrates within French bean leaves (Phaseolus vulgaris L.) for decomposition studies in soils.

    PubMed

    Girardin, Cyril; Rasse, Daniel P; Biron, Philippe; Ghashghaie, Jaleh; Chenu, Claire

    2009-06-01

    The molecular composition of plant residues is suspected to largely govern the fate of their constitutive carbon (C) in soils. Labile compounds, such as metabolic carbohydrates, are affected differently from recalcitrant and structural compounds by soil-C stabilisation mechanisms. Producing (13)C-enriched plant residues with specifically labeled fractions would help us to investigate the fate in soils of the constitutive C of these compounds. The objective of the present research was to test (13)C pulse chase labeling as a method for specifically enriching the metabolic carbohydrate components of plant residues, i.e. soluble sugars and starch. Bean plants were exposed to a (13)CO(2)-enriched atmosphere for 0.5, 1, 2, 3 and 21 h. The major soluble sugars were then determined on water-soluble extracts, and starch on HCl-hydrolysable extracts. The results show a quick differential labeling between water-soluble and water-insoluble compounds. For both groups, (13)C-labeling increased linearly with time. The difference in delta(13)C signature between water-soluble and insoluble fractions was 7 per thousand after 0.5 h and 70 per thousand after 21 h. However, this clear isotopic contrast masked a substantial labeling variability within each fraction. By contrast, metabolic carbohydrates on the one hand (i.e. soluble sugars + starch) and other fractions (essentially cell wall components) on the other hand displayed quite homogeneous signatures within fractions, and a significant difference in labeling between fractions: delta(13)C = 414 +/- 3.7 per thousand and 56 +/- 5.5 per thousand, respectively. Thus, the technique generates labeled plant residues displaying contrasting (13)C-isotopic signatures between metabolic carbohydrates and other compounds, with homogenous signatures within each group. Metabolic carbohydrates being labile compounds, our findings suggest that the technique is particularly appropriate for investigating the effect of compound lability on the long

  19. 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.

  20. Does the time of the sampling matter in 13C pulse labeling and chasing experiments? A case study on beech seedlings

    NASA Astrophysics Data System (ADS)

    Gavrichkova, Olga; Thoms, Ronny; Muhr, Jan; Karlowsky, Stefan; Keitel, Claudia; Kayler, Zachary; Calfapietra, Carlo; Gessler, Arthur; Brugnoli, Enrico; Gleixner, Gerd

    2016-04-01

    13C pulse labeling and chasing is a valuable and very popular tool for determination of the fate and turnover rates of C in plant-soil systems. Continuous isoflux measurements became an accessible reality allowing to cover completely the diurnal variation in label assimilation and respiration fluxes. Label turnover in multiple pools, especially of those located belowground, is more often assessed instead by isolated day-time samplings. By increasing the sampling frequency of belowground compartments we aimed to catch the short-term diurnal variations in label allocation and to link these processes with label dynamics in the aboveground biomass. For these purposes we labeled 3-m height soil-grown European beech seedlings with 13C enriched CO2 and traced the flow of 13C within belowground plant-soil continuum. Continuous soil isoflux measurements were accompanied by a 3-h-frequency sampling of root and soil material during the first 48 h, followed by a daily sampling in the successive 5 days. The amount of label found in microbial biomass depended partially on the amount of roots in the sample. Microbial biomass C (MBC) and microbial respiration showed very strong correlation, suggesting the possibility to use one as a proxy of the other. MBC enrichment showed a clear diurnal pattern with night-time and early morning peaks. These peaks were similar in shape and shifted by one sampling when compared to root sugars enrichment. Soil respiration showed instead a single bell-shape peak in 13C, likely due to a sequence of peaks of root and microbial origin. 13C flow into soil microbial functional groups was assessed less frequently through phospholipid fatty acid analyses (PLFA). The microorganisms were separated into two distinct groups by the time of the appearance of the label in the single PLFAs. The first group was characterized by a fast appearance of the label and higher enrichment and was composed of Gram negative bacteria and saprotrophic fungi likely living in

  1. 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.

  2. Bioconversion of (13)C-labeled microalgal phytosterols to cholesterol by the Northern Bay scallop, Argopecten irradians irradians.

    PubMed

    Giner, José-Luis; Zhao, Hui; Dixon, Mark S; Wikfors, Gary H

    2016-02-01

    Bivalve mollusks lack de novo cholesterol biosynthesis capabilities and therefore rely upon dietary sources of sterols for rapid growth. Microalgae that constitute the main source of nutrition for suspension-feeding bivalves contain a diverse array of phytosterols, in most cases lacking cholesterol. Rapid growth of bivalves on microalgal diets with no cholesterol implies that some phytosterols can satisfy the dietary requirement for cholesterol through metabolic conversion to cholesterol, but such metabolic pathways have not been rigorously demonstrated. In the present study, stable isotope-labeled phytosterols were used to supplement a unialgal diet of Rhodomonas sp. and their biological transformation to cholesterol within scallop tissues was determined using (13)C-NMR spectroscopy. Scallops efficiently dealkylated ∆(5) C29 (24-ethyl) sterols to cholesterol, and the only C28 sterol that was dealkylated efficiently possessed the 24(28)-double bond. Non-metabolized dietary phytosterols accumulated in the soft tissues. Observed formation of ∆(5,7) sterols (provitamin D) from ∆(5) sterols may represent initiation of steroid hormone (possibly ecdysone) biosynthesis. These findings provide a key component necessary for formulation of nutritionally complete microalgal diets for hatchery production of seed for molluscan aquaculture.

  3. 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

  4. 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

  5. Determination of nonylphenol ethoxylates and octylphenol ethoxylates in environmental samples using 13C-labeled surrogate compounds.

    PubMed

    Yoshida, Yasuko; Ito, Azusa; Murakami, Masashi; Murakami, Takayuki; Fujimoto, Hideharu; Takeda, Kikuo; Suzuki, Shigeru; Hori, Masahiro

    2007-10-01

    Alkylphenol polyethoxylates (APEOs) have been widely used as nonionic surfactants in a variety of industrial and commercial products. Typical compounds are nonylphenol polyethoxylates (NPEOs) and octylphenol polyethoxylates (OPEOs), which serve as precursors to nonylphenol (NP) and octylphenol (OP), respectively. NP and 4-t-OP are known to have endocrine disrupting effects on fish (medaka, Oryzias latipes), so it is important to know the concentrations of APEOs in the environment. Because the analytical characteristics of these compounds depend on the length of the ethoxy chain, it is necessary to use appropriate compounds as internal standards or surrogates. We synthesized two 13C-labeled surrogate compounds and used these compounds as internal standards to determine NPEOs and OPEOs by high-performance liquid chromatography (LC)-mass spectrometry. Method detection limits were 0.015 microg/L for NP (2)EO to 0.037 microg/L for NP(12)EO, and 0.011 microg/L for OP(3,6)EO to 0.024 microg/L for OP (4)EO. NPEO concentrations in water from a sewage treatment plant were less than 0.05-0.52 microg/L for final effluent and 1.2-15 microg/L for influent. OPEO concentrations were less than 0.05-0.15 microg/L for the final effluent and less than 0.05-1.1 microg/L for influent. PMID:17972761

  6. 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.

  7. Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

    PubMed Central

    Thakur, Chandar S.; Sama, Jacob N.; Jackson, Melantha E.; Chen, Bin

    2010-01-01

    Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed

  8. A 13C labelling study on carbon fluxes in Arctic plankton communities under elevated CO2 levels

    NASA Astrophysics Data System (ADS)

    de Kluijver, A.; Soetaert, K.; Czerny, J.; Schulz, K. G.; Boxhammer, T.; Riebesell, U.; Middelburg, J. J.

    2012-07-01

    The effect of CO2 on carbon fluxes in Arctic plankton communities was investigated during the 2010 EPOCA mesocosm study in Ny Ålesund, Svalbard. Nine mesocosms were set up with initial pCO2 levels ranging from 185 to 1420 μatm for 5 weeks. 13C labelled bicarbonate was added at the start of the experiment to follow the transfer of carbon from dissolved inorganic carbon (DIC) into phytoplankton, bacteria, total particulate organic carbon (POC), zooplankton, and settling particles. Polar lipid derived fatty acids (PLFA) were used to trace carbon dynamics of phytoplankton and bacteria and allowed distinction of two groups of phytoplankton: phyto I (autotrophs) and phyto II (mixotrophs). Nutrients were added on day 13. A nutrient-phytoplankton-zooplankton-detritus model amended with 13C dynamics was constructed and fitted to the data to quantify uptake rates and carbon fluxes in the plankton community during the phase prior to nutrient addition (phase 1, days 0-12). During the first 12 days, a phytoplankton bloom developed that was characterized by high growth rates (0.87 days-1) for phyto I and lower growth rates (0.18 days-1) for phyto II. A large part of the carbon fixed by phytoplankton (~31%) was transferred to bacteria, while mesozooplankton grazed only ~6% of the production. After 6 days, the bloom collapsed and part of the organic matter subsequently settled into the sediment traps. The sedimentation losses of detritus in phase 1 were low (0.008 days-1) and overall export was only ~7% of production. Zooplankton grazing and detritus sinking losses prior to nutrient addition were sensitive to CO2: grazing decreased with increasing CO2, while sinking increased. Phytoplankton production increased again after nutrient addition on day 13. Although phyto II showed initially higher growth rates with increasing CO2 (days 14-22), the overall production of POC after nutrient addition (phase 2, days 14-29) decreased with increasing CO2. Significant sedimentation occurred

  9. Sensitivity-Enhanced MQ-HCN-CCH-TOCSY and MQ-HCN-CCH-COSY Pulse Schemes for 13C/ 15N Labeled RNA Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Hu, Weidong; Jiang, Licong; Gosser, Yuying Q.

    2000-07-01

    Sensitivity enhanced multiple-quantum 3D HCN-CCH-TOCSY and HCN-CCH-COSY experiments are presented for the ribose resonance assignment of 13C/15N-labeled RNA sample. The experiments make use of the chemical shift dispersion of N1/N9 of pyrimidine/purine to distinguish the ribose spin systems. They provide a complementary approach for the assignment of ribose resonance to the currently used HCCH-COSY and HCCH-TOCSY type experiments in which either 13C or 1H is utilized to separate the different ribose spin systems. The pulse schemes have been demonstrated on a 23-mer 13C/15N-labeled RNA aptamer complexed with neomycin and tested on a 32-mer RNA complexed with a 23-residue peptide.

  10. Synthesis of /sup 13/C-labeled standards for use in coal liquefaction studies. II. Dissolving metal reactions applied to naphthalenes and indoles: effect of sonication

    SciTech Connect

    Pickering, R.E.

    1986-01-01

    High yield syntheses of /sup 13/C-labeled standards for later use in coal liquefaction studies are described. An alternate route for the synthesis of 1-cyclohexanone-1-/sup 13/C is discussed. Naphthalene and 2,3-dimethylnaphthalene were reduced with metal-amine solutions. The effect of sonication on product distribution was investigated. The effect of different amine solvent and different metals also was studied. A series of indoles were reduced with lithium and ethylenediamine to study the effect of sonication. To aid identification of some reduction products, independent syntheses were conducted. Naphthalenol-1-/sup 13/C, phenol-1-/sup 13/C, 1-indanone-1-/sup 13/C, 3,4-dihydro-2(1H)-quinolinone-2-/sup 13/C, 1,2,3,4-tetrahydroquinoline-2-/sup 13/C and carbazole-9a-/sup 13/C were synthesized in good yield. The carbonation of 1,5-pentanedimagnesium bromide was shown to be a viable alternative route for the synthesis of 1-cyclohexanone-1-/sup 13/C. Sonication was found to be an efficient agitation method for the reductive dimerization or the reductive amination of naphthalene and 2,3-dimethylnaphthalene. The product distribution was found to be dependent on the selection of the amine solvent. Sodium was shown to be a better reagent for reductive dimerization than potassium, lithium, calcium, or magnesium. The synthesis of 6,6',7,7'-tetramethyl-,1'-binaphthyl, 6,6',7,7'-tetramethyl-1,2'-binaphthyl, 6,6',7,7'-tetramethyl-2,2'-binaphthyl and some of their hydro-derivatives are described. The reduction of indoles with lithium-ethylenediamine was found to be influenced by sonication which significant increased the yield of the 4,5,6,7-tetrahydro-derivative. N-Substituted indoles were shown to undergo reductive cleavage with lithium-ethylenediamine while being treated with ultrasound.

  11. Expression, purification, and mass spectrometric analysis of 15N, 13C-labeled RGD-hirudin, expressed in Pichia pastoris, for NMR studies.

    PubMed

    Huang, Yinong; Zhang, Yanling; Wu, Yi; Wang, Jue; Liu, Xingang; Dai, Linsen; Wang, Longsheng; Yu, Min; Mo, Wei

    2012-01-01

    A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets. PMID:22879918

  12. INCLUSION OF 13C12-LABELLED MONO-, DI-, AND TRI-CHLORINATED DIBENZO-P-DIOXIN AND DIBENZOFURAN STANDARDS IN U.S. EPA METHODS 0023A/8290

    EPA Science Inventory

    13C12-labeled mono-, di-, and tri-chlorinated dibenzo-p-dioxin (CDD) and -chlorinated dibenzofuran (CDF) standards have been tested for their applicability to standard EPA sampling and analytical Methods 0023A/8290. These methods target for analysis only the tetra- through octa-C...

  13. Changes in microbial structure and functional communities at different soil depths during 13C labelled root litter degradation

    NASA Astrophysics Data System (ADS)

    Sanaullah, Muhammad; Baumann, Karen; Chabbi, Abad; Dignac, Marie-France; Maron, Pierre-Alain; Kuzyakov, Yakov; Rumpel, Cornelia

    2014-05-01

    Soil organic matter turnover depends on substrate quality and microbial activity in soil but little is known about how addition of freshly added organic material modifies the diversity of soil microbial communities with in a soil profile. We took advantage of a decomposition experiment, which was carried out at different soil depths under field conditions and sampled litterbags with 13C-labelled wheat roots, incubated in subsoil horizons at 30, 60 and 90 cm depth for up to 36 months. The effect of root litter addition on microbial community structure, diversity and activity was studied by determining total microbial biomass, PLFA signatures, molecular tools (DNA genotyping and pyrosequencing of 16S and 18S rDNAs) and extracellular enzyme activities. Automated ribosomal intergenic spacer analysis (ARISA) was also carried out to determine the differences in microbial community structure. We found that with the addition of root litter, total microbial biomass as well as microbial community composition and structure changed at different soil depths and change was significantly higher at top 30cm soil layer. Moreover, in the topsoil, population of both gram-positive and gram-negative bacteria increased with root litter addition over time, while subsoil horizons were relatively dominated by fungal community. Extra-cellular enzyme activities confirmed relatively higher fungal community at subsoil horizons compared with surface soil layer with bacteria dominant microbial population. Bacterial-ARISA profiling illustrated that the addition of root litter enhanced the abundance of Actinobacteria and Proteobacteria, at all three soil depths. These bacteria correspond to copiotrophic attributes, which can preferentially consume of labile soil organic C pools. While disappearance of oligotrophic Acidobacteria confirmed the shifting of microbial communities due to the addition of readily available substrate. We concluded that root litter mixing altered microbial community

  14. Using 13C-labeled benzene and Raman gas spectroscopy to investigate respiration and biodegradation kinetics following soil contamination

    NASA Astrophysics Data System (ADS)

    Jochum, Tobias; Popp, Juergen; Frosch, Torsten

    2016-04-01

    Soil and groundwater contamination with benzene can cause serious environmental damages. However, many soil microorganisms are capable to adapt and known to strongly control the fate of organic contamination. Cavity enhanced Raman gas spectroscopy (CERS) was applied to investigate the short-term response of indigenous soil bacteria to a sudden surface contamination with benzene regarding the temporal variations of gas products and their exchange rates with the adjacent atmosphere. 13C-labeled benzene was spiked on a silty-loamy soil column (sampled from Hainich National Park, Germany) in order to track and separate the changes in heterotrophic soil respiration - involving 12CO2 and O2 - from the microbial process of benzene degradation, which ultimately forms 13CO2.1 The respiratory quotient (RQ) of 0.98 decreased significantly after the spiking and increased again within 33 hours to a value of 0.72. This coincided with maximum 13CO2 concentration rates (0.63 μ mol m-2 s-1), indicating highest benzene degradation at 33 hours after the spiking event. The diffusion of benzene in the headspace and the biodegradation into 13CO2 were simultaneously monitored and 12 days after the benzene spiking no measurable degradation was detected anymore.1 The RQ finally returned to a value of 0.96 demonstrating the reestablished aerobic respiration. In summary, this study shows the potential of combining Raman gas spectroscopy and stable isotopes to follow soil microbial biodegradation dynamics while simultaneously monitoring the underlying respiration behavior. Support by the Collaborative Research Center 1076 Aqua Diva is kindly acknowledged. We thank Beate Michalzik for soil analysis and discussion. 1. T. Jochum, B. Michalzik, A. Bachmann, J. Popp and T. Frosch, Analyst, 2015, 140, 3143-3149.

  15. Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

    PubMed

    Nishihara, Tatsuya; Yoshihara, Hikari A I; Nonaka, Hiroshi; Takakusagi, Yoichi; Hyodo, Fuminori; Ichikawa, Kazuhiro; Can, Emine; Bastiaansen, Jessica A M; Takado, Yuhei; Comment, Arnaud; Sando, Shinsuke

    2016-08-26

    The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin. PMID:27483206

  16. 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

  17. 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.

  18. Efficient, scalable and economical preparation of tris(deuterium)- and 13C-labelled N-methyl-N-nitroso-p-toluenesulfonamide (Diazald®) and their conversion to labelled diazomethane.

    PubMed

    Shields, Samuel W J; Manthorpe, Jeffrey M

    2014-10-01

    A method for the preparation of multi-gramme quantities of N-methyl-d3-N-nitroso-p-toluenesulfonamide (Diazald-d3) and N-methyl-(13)C-N-nitroso-p-toluenesulfonamide (Diazald-(13)C) and their conversion to diazomethane-d2 and diazomethane-(13) C, respectively, is presented. This approach uses robust and reliable chemistry, and critically, employs readily commercially available and inexpensive methanol as the label source. Several reactions of labelled diazomethane are also reported, including alkene cyclopropanation, phenol methylation and α-diazoketone formation, as well as deuterium scrambling in the preparation of diazomethane-d2 and subsequent methyl esterification of benzoic acid.

  19. 13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum.

    PubMed

    Kleijn, Roelco J; van Winden, Wouter A; Ras, Cor; van Gulik, Walter M; Schipper, Dick; Heijnen, Joseph J

    2006-07-01

    In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.

  20. Spatial and temporal distribution of 13C labelled plant residues in soil aggregates and Lumbricus terrestris surface casts: A combination of Transmission Electron Microscopy and Nanoscale Secondary Ion Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Vidal, Alix; Remusat, Laurent; Watteau, Françoise; Derenne, Sylvie; Quenea, Katell

    2016-04-01

    Earthworms play a central role in litter decomposition, soil structuration and carbon cycling. They ingest both organic and mineral compounds which are mixed, complexed with mucus and dejected in form of casts at the soil surface and along burrows. Bulk isotopic or biochemical technics have often been used to study the incorporation of litter in soil and casts, but they could not reflect the complex interaction between soil, plant and microorganisms at the microscale. However, the heterogeneous distribution of organic carbon in soil structures induces contrasted microbial activity areas. Nano-scale secondary ion mass spectrometry (NanoSIMS), which is a high spatial resolution method providing elemental and isotopic maps of organic and mineral materials, has recently been applied in soil science (Herrmann et al., 2007; Vogel et al., 2014). The combination of Nano-scale secondary ion mass spectrometry (NanoSIMS) and Transmission Electron Microscopy (TEM) has proven its potential to investigate labelled residues incorporation in earthworm casts (Vidal et al., 2016). In line of this work, we studied the spatial and temporal distribution of plant residues in soil aggregates and earthworm surface casts. This study aimed to (1) identify the decomposition states of labelled plant residues incorporated at different time steps, in casts and soil, (2) identify the microorganisms implied in this decomposition (3) relate the organic matter states of decomposition with their 13C signature. A one year mesocosm experiment was set up to follow the incorporation of 13C labelled Ryegrass (Lolium multiflorum) litter in a soil in the presence of anecic earthworms (Lumbricus terrestris). Soil and surface cast samples were collected after 8 and 54 weeks, embedded in epoxy resin and cut into ultra-thin sections. Soil was fractionated and all and analyzed with TEM and NanoSIMS, obtaining secondary ion images of 12C, 16O, 12C14N, 13C14N and 28Si. The δ13C maps were obtained using the 13C14

  1. 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.

  2. An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in 15N, 13C labeled proteins.

    PubMed

    Mäntylahti, Sampo; Tossavainen, Helena; Hellman, Maarit; Permi, Perttu

    2009-11-01

    An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely (13)C'(i), (15)N(i), (1)HN(i) connectivities in uniformly 15N/13C-labeled proteins. In comparison to the "out-and-back" style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the (13)C' dimension and on average 1.6 times higher sensitivity especially for residues in alpha-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(HCA)CO(CA)NH experiment in combination with the established HNCO experiment was crucial for the assignment of highly disordered CT16. PMID:19768387

  3. 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

  4. Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate.

    PubMed Central

    Thompson, J E; Smith, R C; Fry, S C

    1997-01-01

    Xyloglucan from the walls of Rosa cells that had been cultured on [12C]- or [13C]-glucose formed bands in caesium trifluoroacetate with mean buoyant densities of 1.575 or 1.616 g/ml respectively. Incubation of a mixture of [13C,3H]xyloglucan and [12C,1H]xyloglucan in the presence of xyloglucan endotransglycosylase (XET) activity caused the mean buoyant density of the radioactive material to decrease, indicating that interpolymeric transglycosylation could be detected in vitro. We used two 13C/3H-dual-labelling protocols to look for interpolymeric transglycosylation in vivo. In protocol A, [13C]glucose-grown Rosa cells were transferred into [12C]glucose medium 6 h after a approximately 2 h pulse of l-[1-3H]arabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound [3H]xyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized [12C,1H]xyloglucan, it became covalently attached to previously wall-bound [13C, 3H]xyloglucan. In protocol B, [12C]glycerol- or [12C]glucose-grown Rosa cells were transferred into [13C]glucose medium, 20 or 60 min before a approximately 2 h pulse of [3H]arabinose. The buoyant density of the earliest wall-bound [3H]xyloglucan showed that it had a 12C/13C ratio of approximately 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized [13C, 3H]xyloglucan became covalently attached to previously synthesized [12C]xyloglucan. During the following 7 days in culture, the mean buoyant density of the [3H]xyloglucan increased, showing that later-synthesized [13C,1H]xyloglucan can be covalently attached to previously wall-bound [12C,13C,3H]xyloglucan. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET. We conclude that XET-catalysed interpolymeric transglycosylation

  5. Effects of growth and tissue type on the kinetics of 13C and 15N incorporation in a rapidly growing ectotherm.

    PubMed

    Reich, Kimberly J; Bjorndal, Karen A; Martínez Del Rio, Carlos

    2008-04-01

    The use of stable isotopes to investigate animal diets, habitat use, and trophic level requires understanding the rate at which animals incorporate the 13C and 15N from their diets and the factors that determine the magnitude of the difference in isotopic composition between the animal's diet and that of its tissues. We determined the contribution of growth and catabolic turnover to the rate of 13C and 15N incorporation into several tissues that can be sampled non-invasively (skin, scute, whole blood, red blood cells, and plasma solutes) in two age classes of a rapidly growing ectotherm (loggerhead turtles, Caretta caretta). We found significant differences in C and N incorporation rates and isotopic discrimination factors (Delta 13C = delta 13Ctissues - delta 13Cdiet and Delta 15N = delta 15Ntissues - delta 15Ndiet) among tissues and between age classes. Growth explained from 26 to 100% of the total rate of incorporation in hatchling turtles and from 15 to 52% of the total rate of incorporation in juvenile turtles. Because growth contributed significantly to the rate of isotopic incorporation, variation in rates among tissues was lower than reported in previous studies. The contribution of growth can homogenize the rate of isotopic incorporation and limit the application of stable isotopes to identify dietary changes at contrasting time scales and to determine the timing of diet shifts. The isotopic discrimination factor of nitrogen ranged from -0.64 to 1.77 per thousand in the turtles' tissues. These values are lower than the commonly assumed average 3.4 per thousand discrimination factors reported for whole body and muscle isotopic analyses. The increasing reliance on non-invasive and non-destructive sampling in animal isotopic ecology requires that we recognize and understand why different tissues differ in isotopic discrimination factors.

  6. Evidence of polycyclic aromatic hydrocarbon biodegradation in a contaminated aquifer by combined application of in situ and laboratory microcosms using (13)C-labelled target compounds.

    PubMed

    Bahr, Arne; Fischer, Anko; Vogt, Carsten; Bombach, Petra

    2015-02-01

    The number of approaches to evaluate the biodegradation of polycyclic aromatic hydrocarbons (PAHs) within contaminated aquifers is limited. Here, we demonstrate the applicability of a novel method based on the combination of in situ and laboratory microcosms using (13)C-labelled PAHs as tracer compounds. The biodegradation of four PAHs (naphthalene, fluorene, phenanthrene, and acenaphthene) was investigated in an oxic aquifer at the site of a former gas plant. In situ biodegradation of naphthalene and fluorene was demonstrated using in situ microcosms (BACTRAP(®)s). BACTRAP(®)s amended with either [(13)C6]-naphthalene or [(13)C5/(13)C6]-fluorene (50:50) were incubated for a period of over two months in two groundwater wells located at the contaminant source and plume fringe, respectively. Amino acids extracted from BACTRAP(®)-grown cells showed significant (13)C-enrichments with (13)C-fractions of up to 30.4% for naphthalene and 3.8% for fluorene, thus providing evidence for the in situ biodegradation and assimilation of those PAHs at the field site. To quantify the mineralisation of PAHs, laboratory microcosms were set up with BACTRAP(®)-grown cells and groundwater. Naphthalene, fluorene, phenanthrene, or acenaphthene were added as (13)C-labelled substrates. (13)C-enrichment of the produced CO2 revealed mineralisation of between 5.9% and 19.7% for fluorene, between 11.1% and 35.1% for acenaphthene, between 14.2% and 33.1% for phenanthrene, and up to 37.0% for naphthalene over a period of 62 days. Observed PAH mineralisation rates ranged between 17 μg L(-1) d(-1) and 1639 μg L(-1) d(-1). The novel approach combining in situ and laboratory microcosms allowed a comprehensive evaluation of PAH biodegradation at the investigated field site, revealing the method's potential for the assessment of PAH degradation within contaminated aquifers.

  7. Development of an LC-MS/MS method for the determination of endogenous cortisol in hair using (13)C3-labeled cortisol as surrogate analyte.

    PubMed

    Binz, Tina M; Braun, Ueli; Baumgartner, Markus R; Kraemer, Thomas

    2016-10-15

    Hair cortisol levels are increasingly applied as a measure for stress in humans and mammals. Cortisol is an endogenous compound and is always present within the hair matrix. Therefore, "cortisol-free hair matrix" is a critical point for any analytical method to accurately quantify especially low cortisol levels. The aim of this project was to modify current methods used for hair cortisol analysis to more accurately determine low endogenous cortisol concentrations in hair. For that purpose, (13)C3-labeled cortisol, which is not naturally present in hair (above 13C natural abundance levels), was used for calibration and comparative validation applying cortisol versus (13)C3-labeled cortisol. Cortisol was extracted from 20mg hair (standard sample amount) applying an optimized single step extraction protocol. An LC-MS/MS method was developed for the quantitative analysis of cortisol using either cortisol or (13)C3-cortisol as calibrators and D7-cortisone as internal standard (IS). The two methods (cortisol/(13)C3-labeled cortisol) were validated in a concentration range up to 500pg/mg and showed good linearity for both analytes (cortisol: R(2)=0.9995; (13)C3-cortisol R(2)=0.9992). Slight differences were observed for limit of detection (LOD) (0.2pg/mg/0.1pg/mg) and limit of quantification (LOQ) (1pg/mg/0.5pg/mg). Precision was good with a maximum deviation of 8.8% and 10% for cortisol and (13)C3-cortisol respectively. Accuracy and matrix effects were good for both analytes except for the quality control (QC) low cortisol. QC low (2.5pg/mg) showed matrix effects (126.5%, RSD 35.5%) and accuracy showed a deviation of 26% when using cortisol to spike. These effects are likely to be caused by the unknown amount of endogenous cortisol in the different hair samples used to determine validation parameters like matrix effect, LOQ and accuracy. No matrix effects were observed for the high QC (400pg/mg) samples. Recovery was good with 92.7%/87.3% (RSD 9.9%/6.2%) for QC low and

  8. Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy.

    PubMed

    Flögel, U; Willker, W; Leibfritz, D

    1997-04-01

    13C nuclear magnetic resonance spectroscopy was used to determine the absolute amounts to de novo synthesized amino acids in both the perchloric acid extracts and the hydrolyzed protein fractions of F98 glioma cells incubated for 2 h with 5 mmol/l [U-13C]glucose. 13C NMR spectra of the hydrolyzed protein fraction revealed a marked incorporation of 13C-labelled alanine, aspartate and glutamate into the proteins of F98 cells within the incubation period. Additionally, small amounts of 13C-labelled glycine, proline and serine could unambiguously be identified in the protein fraction. Astonishingly, approximately equal amounts of 13C-labelled glutamate and aspartate were incorporated into the cellular proteins, although the cytosolic steady-state concentration of aspartate was below 13C NMR detectability. Hypertonic stress decreased the incorporation of 13C-labelled amino acids into the total protein, albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. On the other hand, hypotonic stress increased the amount of 13C-labelled proline incorporated into the cellular proteins even though the cytosolic concentration of 13C-labelled proline was largely decreased. Apparently, hypoosmotic conditions stimulate the synthesis of proteins or peptides with a high proline content. The results show that already after 2 h of incubation with [U-13C]glucose there is a pronounced flux of 13C label into the cellular proteins, which is usually disregarded if cytosolic fluids are examined only. This means that calculations of metabolic fluxes based on 13C NMR spectroscopic data obtained from perchloric acid extracts of cells or tissues and also from in vivo measurements consider only the labelled 'NMR visible' cytosolic metabolites, which may have to be corrected for fast label flowing off into other compartments.

  9. A hypersaline microbial mat from the Pacific Atoll Kiritimati: insights into composition and carbon fixation using biomarker analyses and a 13C-labeling approach.

    PubMed

    Bühring, S I; Smittenberg, R H; Sachse, D; Lipp, J S; Golubic, S; Sachs, J P; Hinrichs, K-U; Summons, R E

    2009-06-01

    Modern microbial mats are widely recognized as useful analogs for the study of biogeochemical processes relevant to paleoenvironmental reconstruction in the Precambrian. We combined microscopic observations and investigations of biomarker composition to investigate community structure and function in the upper layers of a thick phototrophic microbial mat system from a hypersaline lake on Kiritimati (Christmas Island) in the Northern Line Islands, Republic of Kiribati. In particular, an exploratory incubation experiment with (13)C-labeled bicarbonate was conducted to pinpoint biomarkers from organisms actively fixing carbon. A high relative abundance of the cyanobacterial taxa Aphanocapsa and Aphanothece was revealed by microscopic observation, and cyanobacterial fatty acids and hydrocarbons showed (13)C-uptake in the labeling experiment. Microscopic observations also revealed purple sulfur bacteria (PSB) in the deeper layers. A cyclic C(19:0) fatty acid and farnesol were attributed to this group that was also actively fixing carbon. Background isotopic values indicate Calvin-Benson cycle-based autotrophy for cycC(19:0) and farnesol-producing PSBs. Biomarkers from sulfate-reducing bacteria (SRB) in the top layer of the mat and their (13)C-uptake patterns indicated a close coupling between SRBs and cyanobacteria. Archaeol, possibly from methanogens, was detected in all layers and was especially abundant near the surface where it contained substantial amounts of (13)C-label. Intact glycosidic tetraether lipids detected in the deepest layer indicated other archaea. Large amounts of ornithine and betaine bearing intact polar lipids could be an indicator of a phosphate-limited ecosystem, where organisms that are able to substitute these for phospholipids may have a competitive advantage.

  10. A hypersaline microbial mat from the Pacific Atoll Kiritimati: insights into composition and carbon fixation using biomarker analyses and a 13C-labeling approach.

    PubMed

    Bühring, S I; Smittenberg, R H; Sachse, D; Lipp, J S; Golubic, S; Sachs, J P; Hinrichs, K-U; Summons, R E

    2009-06-01

    Modern microbial mats are widely recognized as useful analogs for the study of biogeochemical processes relevant to paleoenvironmental reconstruction in the Precambrian. We combined microscopic observations and investigations of biomarker composition to investigate community structure and function in the upper layers of a thick phototrophic microbial mat system from a hypersaline lake on Kiritimati (Christmas Island) in the Northern Line Islands, Republic of Kiribati. In particular, an exploratory incubation experiment with (13)C-labeled bicarbonate was conducted to pinpoint biomarkers from organisms actively fixing carbon. A high relative abundance of the cyanobacterial taxa Aphanocapsa and Aphanothece was revealed by microscopic observation, and cyanobacterial fatty acids and hydrocarbons showed (13)C-uptake in the labeling experiment. Microscopic observations also revealed purple sulfur bacteria (PSB) in the deeper layers. A cyclic C(19:0) fatty acid and farnesol were attributed to this group that was also actively fixing carbon. Background isotopic values indicate Calvin-Benson cycle-based autotrophy for cycC(19:0) and farnesol-producing PSBs. Biomarkers from sulfate-reducing bacteria (SRB) in the top layer of the mat and their (13)C-uptake patterns indicated a close coupling between SRBs and cyanobacteria. Archaeol, possibly from methanogens, was detected in all layers and was especially abundant near the surface where it contained substantial amounts of (13)C-label. Intact glycosidic tetraether lipids detected in the deepest layer indicated other archaea. Large amounts of ornithine and betaine bearing intact polar lipids could be an indicator of a phosphate-limited ecosystem, where organisms that are able to substitute these for phospholipids may have a competitive advantage. PMID:19476506

  11. Elucidating metabolic pathways for amino acid incorporation into dragline spider silk using 13C enrichment and solid state NMR.

    PubMed

    Creager, Melinda S; Izdebski, Thomas; Brooks, Amanda E; Lewis, Randolph V

    2011-07-01

    Spider silk has been evolutionarily optimized for contextual mechanical performance over the last 400 Ma. Despite precisely balanced mechanical properties, which have yet to be reproduced, the underlying molecular architecture of major ampullate spider silk can be simplified being viewed as a versatile block copolymer. Four primary amino acid motifs: polyalanine, (GA)(n), GPGXX, and GGX (X = G,A,S,Q,L,Y) will be considered in this study. Although synthetic mimetics of many of these amino acid motifs have been produced in several biological systems, the source of spider silk's mechanical integrity remains elusive. Mechanical robustness may be a product not only of the amino acid structure but also of the tertiary structure of the silk. Historically, solid state nuclear magnetic resonance (ssNMR) has been used to reveal the crystalline structure of the polyalanine motif; however, limitations in amino acid labeling techniques have obscured the structures of the GGX and GPGXX motifs thought to be responsible for the structural mobility of spider silk. We describe the use of metabolic pathways to label tyrosine for the first time as well as to improve the labeling efficiency of proline. These improved labeling techniques will allow the previously unknown tertiary structures of major ampullate silk to be probed. PMID:21334448

  12. Multidimensional High-Resolution Magic Angle Spinning and Solution-State NMR Characterization of (13)C-labeled Plant Metabolites and Lignocellulose.

    PubMed

    Mori, Tetsuya; Tsuboi, Yuuri; Ishida, Nobuhiro; Nishikubo, Nobuyuki; Demura, Taku; Kikuchi, Jun

    2015-01-01

    Lignocellulose, which includes mainly cellulose, hemicellulose, and lignin, is a potential resource for the production of chemicals and for other applications. For effective production of materials derived from biomass, it is important to characterize the metabolites and polymeric components of the biomass. Nuclear magnetic resonance (NMR) spectroscopy has been used to identify biomass components; however, the NMR spectra of metabolites and lignocellulose components are ambiguously assigned in many cases due to overlapping chemical shift peaks. Using our (13)C-labeling technique in higher plants such as poplar samples, we demonstrated that overlapping peaks could be resolved by three-dimensional NMR experiments to more accurately assign chemical shifts compared with two-dimensional NMR measurements. Metabolites of the (13)C-poplar were measured by high-resolution magic angle spinning NMR spectroscopy, which allows sample analysis without solvent extraction, while lignocellulose components of the (13)C-poplar dissolved in dimethylsulfoxide/pyridine solvent were analyzed by solution-state NMR techniques. Using these methods, we were able to unambiguously assign chemical shifts of small and macromolecular components in (13)C-poplar samples. Furthermore, using samples of less than 5 mg, we could differentiate between two kinds of genes that were overexpressed in poplar samples, which produced clearly modified plant cell wall components. PMID:26143886

  13. Evaluation of biodegradability of phenol and bisphenol A during mesophilic and thermophilic municipal solid waste anaerobic digestion using 13C-labeled contaminants.

    PubMed

    Limam, Intissar; Mezni, Mohamed; Guenne, Angéline; Madigou, Céline; Driss, Mohamed Ridha; Bouchez, Théodore; Mazéas, Laurent

    2013-01-01

    In this paper, the isotopic tracing using (13)C-labeled phenol and bisphenol A was used to study their biodegradation during anaerobic digestion of municipal solid waste. Microcosms were incubated anaerobically at 35 °C (mesophilic conditions) and 55 °C (thermophilic conditions) without steering. A continuous follow-up of the production of biogas (CH(4) and CO(2)), was carried out during 130 d until the establishment of stable methanogenesis. Then (13)C(12)-BPA, and (13)C(6)-phenol were injected in microcosms and the follow-up of their degradation was performed simultaneously by gas chromatography isotope-ratio mass spectrometry (GC-IRMS) and gas chromatography mass spectrometry (GC-MS). Moreover, Carbon-13 Nuclear Magnetic Resonance ((13)C-NMR) Spectroscopy is used in the identification of metabolites. This study proves that the mineralization of phenol to CO(2) and CH(4) occurs during anaerobic digestion both in mesophilic and thermophilic conditions with similar kinetics. In mesophilic condition phenol degradation occurs through the benzoic acid pathway. In thermophilic condition it was not possible to identify the complete metabolic pathway as only acetate was identified as metabolite. Our results suggest that mineralization of phenol under thermophilic condition is instantaneous explaining why metabolites are not observed as they do not accumulate. No biodegradation of BPA was observed.

  14. Multidimensional High-Resolution Magic Angle Spinning and Solution-State NMR Characterization of 13C-labeled Plant Metabolites and Lignocellulose

    PubMed Central

    Mori, Tetsuya; Tsuboi, Yuuri; Ishida, Nobuhiro; Nishikubo, Nobuyuki; Demura, Taku; Kikuchi, Jun

    2015-01-01

    Lignocellulose, which includes mainly cellulose, hemicellulose, and lignin, is a potential resource for the production of chemicals and for other applications. For effective production of materials derived from biomass, it is important to characterize the metabolites and polymeric components of the biomass. Nuclear magnetic resonance (NMR) spectroscopy has been used to identify biomass components; however, the NMR spectra of metabolites and lignocellulose components are ambiguously assigned in many cases due to overlapping chemical shift peaks. Using our 13C-labeling technique in higher plants such as poplar samples, we demonstrated that overlapping peaks could be resolved by three-dimensional NMR experiments to more accurately assign chemical shifts compared with two-dimensional NMR measurements. Metabolites of the 13C-poplar were measured by high-resolution magic angle spinning NMR spectroscopy, which allows sample analysis without solvent extraction, while lignocellulose components of the 13C-poplar dissolved in dimethylsulfoxide/pyridine solvent were analyzed by solution-state NMR techniques. Using these methods, we were able to unambiguously assign chemical shifts of small and macromolecular components in 13C-poplar samples. Furthermore, using samples of less than 5 mg, we could differentiate between two kinds of genes that were overexpressed in poplar samples, which produced clearly modified plant cell wall components. PMID:26143886

  15. HNCA-TOCSY-CANH experiments with alternate 13C-12C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

    PubMed Central

    Takeuchi, Koh; Gal, Maayan; Takahashi, Hideo; Shimada, Ichio

    2011-01-01

    Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak 3JCαCα coupling. These pulse sequences, which resemble recently described 13C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in 1H2O, and use 1H excitation and detection. These experiments require alternate 13C-12C labeling together with perdeuteration, which allows utilizing the small 3JCαCα scalar coupling that is otherwise masked by the stronger 1JCC couplings in uniformly 13C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the 13Cα of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOC-SY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the 15N-1H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments. PMID:21110064

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

    PubMed

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

    2015-01-01

    Knowledge of how ribonucleic acid (RNA) structures fold to form intricate, three-dimensional structures has provided fundamental insights into understanding the biological functions of RNA. Nuclear magnetic resonance (NMR) spectroscopy is a particularly useful high-resolution technique to investigate the dynamic structure of RNA. Effective study of RNA by NMR requires enrichment with isotopes of (13)C or (15)N or both. Here, we present a method to produce milligram quantities of uniformly (15)N- and site-specifically (13)C-labeled RNAs using wild-type K12 and mutant tktA Escherichia coli in combination with a tRNA-scaffold approach. The method includes a double selection protocol to obtain an E. coli clone with consistently high expression of the recombinant tRNA-scaffold. We also present protocols for the purification of the tRNA-scaffold from a total cellular RNA extract and the excision of the RNA of interest from the tRNA-scaffold using DNAzymes. Finally, we showcase NMR applications to demonstrate the benefit of using in vivo site-specifically (13)C-labeled RNA. PMID:26577743

  17. Timing and magnitude of C partitioning through a young loblolly pine (Pinus taeda L.) stand using 13C labeling and shade treatments

    DOE PAGES

    Warren, Jeffrey M.; Iversen, Colleen M.; Garten, Jr., Charles T.; Norby, Richard J.; Childs, Joanne; Brice, Deanne Jane; Evans, R. M.; Gu, Lianhong; Thornton, Peter E.; Weston, David J.

    2011-12-30

    The dynamics of rapid changes in carbon (C) partitioning within forest ecosystems are not well understood, which limits improvement of mechanistic models of C cycling. Our objective was to inform model processes by describing relationships between C partitioning and accessible environmental or physiological measurements, with a special emphasis on short-term C flux through a forest ecosystem. We exposed eight 7-year-old loblolly pine (Pinus taeda L.) trees to air enriched with 13CO2 and then implemented adjacent light shade (LS) and heavy shade (HS) treatments in order to manipulate C uptake and flux. The impacts of shading on photosynthesis, plant water potential,more » sap flow, basal area growth, root growth, and soil CO2 efflux rate (CER) were assessed for each tree over a three-week period. The progression of the 13C label was concurrently tracked from the atmosphere through foliage, phloem, roots, and surface soil CO2 efflux. The HS treatment significantly reduced C uptake, sap flow, stem growth and fine root standing crop, and resulted in greater residual soil water content to 1 m depth. Sap flow was strongly correlated with CER on the previous day, but not the current day, with no apparent treatment effect on the relationship. Although there were apparent reductions in new C flux belowground, the heavy shade treatment did not noticeably reduce the magnitude of belowground autotrophic and heterotrophic respiration based on surface soil CO2 efflux rate (CER), which was overwhelmingly driven by soil temperature and moisture. The 13C label was immediately detected in foliage on label day (half-life = 0.5 d), progressed through phloem by day 2 (half-life = 4.7 d), roots by day 2-4, and subsequently was evident as respiratory release from soil which peaked between days 3-6. The δ13C of soil CO2 efflux was strongly correlated with phloem 13C on the previous day, or two days earlier. While the 13C label was readily tracked through the ecosystem, the fate of root

  18. Comparison of different mass spectrometry techniques in the measurement of L-[ring-(13)C6]phenylalanine incorporation into mixed muscle proteins.

    PubMed

    Zabielski, Piotr; Ford, G Charles; Persson, X Mai; Jaleel, Abdul; Dewey, Jerry D; Nair, K Sreekumaran

    2013-02-01

    Precise measurement of low enrichment of stable isotope labeled amino-acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L-[ring-(13)C(6)]phenylalanine and a bolus dose of L-[ring-(13)C(6)]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC-combustion-isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring-(13)C(6)]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R(2)= 0.9962 and R(2) = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra-assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter-assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L-[ring-(13)C(6)]phenylalanine tracer enrichment in low abundance and in small quantity samples. PMID:23378099

  19. Comparison of different mass spectrometry techniques in the measurement of L-[ring-(13)C6]phenylalanine incorporation into mixed muscle proteins.

    PubMed

    Zabielski, Piotr; Ford, G Charles; Persson, X Mai; Jaleel, Abdul; Dewey, Jerry D; Nair, K Sreekumaran

    2013-02-01

    Precise measurement of low enrichment of stable isotope labeled amino-acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L-[ring-(13)C(6)]phenylalanine and a bolus dose of L-[ring-(13)C(6)]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC-combustion-isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring-(13)C(6)]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R(2)= 0.9962 and R(2) = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra-assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter-assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L-[ring-(13)C(6)]phenylalanine tracer enrichment in low abundance and in small quantity samples.

  20. Quantitation of a spin polarization-induced nuclear Overhauser effect (SPINOE) between a hyperpolarized 13C-labeled cell metabolite and water protons

    PubMed Central

    Marco-Rius, Irene; Bohndiek, Sarah E; Kettunen, Mikko I; Larkin, Timothy J; Basharat, Meer; Seeley, Colm; Brindle, Kevin M

    2014-01-01

    The spin polarization-induced nuclear Overhauser effect (SPINOE) describes the enhancement of spin polarization of solvent nuclei by the hyperpolarized spins of a solute. In this communication we demonstrate that SPINOEs can be observed between [1,4-13C2]fumarate, hyperpolarized using the dissolution dynamic nuclear polarization technique, and solvent water protons. We derive a theoretical expression for the expected enhancement and demonstrate that this fits well with experimental measurements. Although the magnitude of the effect is relatively small (around 2% measured here), the SPINOE increases at lower field strengths, so that at clinically relevant magnetic fields (1.5–3 T) it may be possible to track the passage through the circulation of a bolus containing a hyperpolarized 13C-labeled substrate through the increase in solvent water 1H signal. © 2014 The Authors. Contrast Media & Molecular Imaging published by John Wiley and Sons, Ltd. PMID:24523064

  1. Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.

    PubMed

    Fasshuber, Hannes Klaus; Demers, Jean-Philippe; Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

    2015-03-01

    Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively (13)C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

  2. Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies

    NASA Astrophysics Data System (ADS)

    Fasshuber, Hannes Klaus; Demers, Jean-Philippe; Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

    2015-03-01

    Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively 13C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

  3. Simultaneous quantification of labeled (2)H5-glycerol, (13)C6-glucose, and endogenous D-glucose in mouse plasma using liquid chromatography tandem mass spectrometry.

    PubMed

    Jahouh, Farid; Wang, Rong

    2015-11-01

    Monitoring the level of glucose and glycerol or their labeled derivatives in biological fluid for kinetic studies has always been challenging, especially in mice, because of the limited volume in addition to the complexity of plasma. For such application, we developed a simple, fast, and sensitive method for the simultaneous measurement of absolute concentrations of labeled (2)H5-glycerol and (13)C6-glucose as well as endogenous D-glucose using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In our study, 15.0 μL of mouse plasma was processed by a one-step protein precipitation, followed by LC-MS/MS analysis. The quantification of the analytes was carried out by monitoring the product ion scan of their corresponding deprotonated molecular ions and constructing the extracted ion fragmentogram by choosing a specific product ion for each analyte (equivalent to precursor ion to product ion transitions). The limit of detection (LOD) was evaluated to be 1.0 μM for both (2)H5-glycerol and (13)C6-glucose, and the limit of quantitation (LOQ) was observed to be 5.0 μM for both (2)H5-glycerol and (13)C6-glucose in diluted mice plasma that corresponds to 50 μM in plasma or 4.60 and 9.01 mg/dL of glycerol and glucose in plasma, respectively. The extraction recoveries are 81.9 % (CV = 8.1 %) for (2)H5-glycerol and 26.2 % (CV = 13.6 %) for (13)C6-glucose. PMID:26362155

  4. 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.

  5. Dipolar-coupling-mediated total correlation spectroscopy in solid-state 13C NMR: Selection of individual 13C- 13C dipolar interactions

    NASA Astrophysics Data System (ADS)

    Spano, Justin; Wi, Sungsool

    2010-06-01

    Herein is described a useful approach in solid-state NMR, for selecting homonuclear 13C- 13C spin pairs in a multiple- 13C homonuclear dipolar coupled spin system. This method builds upon the zero-quantum (ZQ) dipolar recoupling method introduced by Levitt and coworkers (Marin-Montesinos et al., 2006 [30]) by extending the originally introduced one-dimensional (1D) experiment into a two-dimensional (2D) method with selective irradiation scheme, while moving the 13C- 13C mixing scheme from the transverse to the longitudinal mode, together with a dramatic improvement in the proton decoupling efficiency. Selective spin-pair recoupling experiments incorporating Gaussian and cosine-modulated Gaussian pulses for inverting specific spins were performed, demonstrating the ability to detect informative, simplified/individualized, long-range 13C- 13C homonuclear dipolar coupling interactions more accurately by removing less informative, stronger, short-range 13C- 13C interactions from 2D correlation spectra. The capability of this new approach was demonstrated experimentally on uniformly 13C-labeled Glutamine and a tripeptide sample, GAL.

  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. 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

  8. Multidimensional solid-state NMR studies of the structure and dynamics of pectic polysaccharides in uniformly 13C-labeled Arabidopsis primary cell walls

    SciTech Connect

    Dick-Perez, Marilu; Wang, Tuo; Salazar, Andre; Zabotina, Olga A.; Hong, Mei

    2012-07-08

    Plant cell wall (CW) polysaccharides are responsible for the mechanical strength and growth of plant cells; however, the high-resolution structure and dynamics of the CW polysaccharides are still poorly understood because of the insoluble nature of these molecules. Here, we use 2D and 3D magic-angle-spinning (MAS) solid-state NMR (SSNMR) to investigate the structural role of pectins in the plant CW. Intact and partially depectinated primary CWs of Arabidopsis thaliana were uniformly labeled with 13C and their NMR spectra were compared. Recent 13C resonance assignment of the major polysaccharides in Arabidopsis thaliana CWs allowed us to determine the effects of depectination on the intermolecular packing and dynamics of the remaining wall polysaccharides. 2D and 3D correlation spectra show the suppression of pectin signals, confirming partial pectin removal by chelating agents and sodium carbonate. Importantly, higher cross peaks are observed in 2D and 3D 13C spectra of the depectinated CW, suggesting higher rigidity and denser packing of the remaining wall polysaccharides compared with the intact CW. 13C spin–lattice relaxation times and 1H rotating-frame spin–lattice relaxation times indicate that the polysaccharides are more rigid on both the nanosecond and microsecond timescales in the depectinated CW. Taken together, these results indicate that pectic polysaccharides are highly dynamic and endow the polysaccharide network of the primary CW with mobility and flexibility, which may be important for pectin functions. This study demonstrates the capability of multidimensional SSNMR to determine the intermolecular interactions and dynamic structures of complex plant materials under near-native conditions. Copyright © 2012 John Wiley & Sons, Ltd.

  9. hnCOcaNH and hncoCANH pulse sequences for rapid and unambiguous backbone assignment in (13C, 15N) labeled proteins.

    PubMed

    Kumar, Dinesh; Reddy, Jithender G; Hosur, Ramakrishna V

    2010-09-01

    Time-saving in data acquisition is a major thrust of NMR pulse sequence development in the context of structural proteomics research. The conventional HNCA and HN(CA)CO pulse sequences, routinely used for sequential backbone assignment, have the limitation that they cannot distinguish inter- and intra-residue correlations. In order to remove this ambiguity, one has to record HNCO and HN(CO)CA or sequential HNCA experiments which provide unambiguous information of sequential correlations. However, this almost doubles the experimental time. Besides, they require repeated scanning through the (15)N planes to search for the matching peaks along the carbon dimension. In this background, we present here two pulse sequences, termed as hncoCANH and hnCOcaNH that lead to spectra equivalent to HNCA and HN(CA)CO spectra, respectively, but with direct distinction of inter- and intra-residue peaks; these occur with opposite signs in the new experiments. The two pulse sequences have been derived by simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] to frequency-label (13)C(alpha) or (13)C' instead of (15)N during the t(1) period. Like HN(C)N, these spectra also exhibit special patterns of self and sequential peaks around glycines and prolines, which enable direct identification of certain triplets of residues and thus provide internal checks during the sequential assignment walk. The spectra enable rapid and unambiguous assignment of H(N), (15)N and (13)C(alpha) (or (13)C') in a single experiment, and thus would be of great value in high-throughput structural proteomics. PMID:20643567

  10. 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...

  11. Studies of nitrogen metabolism using /sup 13/C NMR spectroscopy. 3. Synthesis of DL-(3-/sup 13/C,2-/sup 15/N)Lysine and its incorporation into streptothricin F/sup 1/

    SciTech Connect

    Gould, S.J.; Thiruvengadam, T.K.

    1981-11-04

    A scheme for the synthesis of DL-(3-/sup 13/C, 2-/sup 15/N)Lysine, I, is presented. Data are also reported to show that the mutase reaction occurring in the biosynthesis of I occurs with an intramolecular migration of nitrogen from C-2 to C-3. (BLM)

  12. Nuclear magnetic resonance study of interaction of ligands with Streptococcus faecium dihydrofolate reductase labeled with (. gamma. -/sup 13/C)tryptophan

    SciTech Connect

    London, R.E.; Groff, J.P.; Cocco, L.; Blakley, R.L.

    1982-01-01

    Dihydrofolate reductase from Streptococcus faecium has been labeled with (..gamma..-/sup 13/C)tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the /sup 13/C NMR spectrum of the labeled enzyme, due to its interaction with various ligands. These include the coenzyme, NPDPH and related nucleotides, folate and its polyglutamate derivatives, and many inhibitors including methotrexate and trimethoprim. In addition, paramagnetic relaxation effects produced by a bound spin-labeled analogue of 2'-phosphoadenosine-5'-diphosphoribose on the tryptophan C/sup ..gamma../ carbons have been measured. Distances calculated from the relaxation data have been compared with corresponding distances in the crystallographic model of the NADPH-methotrexate ternary complex of Lactobacillus casei reductase. The paramagnetic relaxation data indicate that the two downfield resonances (1 and 2) correspond to tryptophans (W/sub A/ and W/sub B/) that are more remote from the catalytic site, and from the crystallographic model these are seen to be Trp-115 and Trp-160. The upfield resonances (3 and 4) that show broadening due to chemical exchange correspond to closer residues (W/sub C/ and W/sub D/), and these are identified with Trp-6 and Trp-22. However, the relaxation data do not permit specific assignments within the nearer and farther pairs. Although resonance 3, which is split due to chemical exchange, was formerly assigned to Trp-6, data obtained for the enzyme in the presence of various ligands are better interpreted if resonance 3 is assigned to Trp-22, which is located on a loop that joins elements of secondary structure and forms one side of the ligand-binding cavity.

  13. Synthesis of [13C4]-labeled ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol as internal standards for reducing ion suppressing/alteration effects in LC/MS-MS quantification.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2014-09-01

    (-)-∆9-Tetrahydrocannabinol is the principal psychoactive component of the cannabis plant and also the active ingredient in some prescribed drugs. To detect and control misuse and monitor administration in clinical settings, reference samples of the native drugs and their metabolites are needed. The accuracy of liquid chromatography/mass spectrometric quantification of drugs in biological samples depends among others on ion suppressing/alteration effects. Especially, 13C-labeled drug analogues are useful for minimzing such interferences. Thus, to provide internal standards for more accurate quantification and for identification purpose, synthesis of [13C4]-∆9-tetrahydro-cannabinol and [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was developed via [13C4]-olivetol. Starting from [13C4]-olivetol the synthesis of [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was shortened from three to two steps by employing nitromethane as a co-solvent in condensation with (+)-apoverbenone.

  14. Carbon sequestration and estimated carbon credit values as measured using 13C labelling and analysis by means of an optical breath test analyser.

    PubMed

    Hood, R C; Khan, M; Haque, A; Khadir, M; Bonetto, J P; Syamsul, R; Mayr, L; Heiling, M

    2004-05-01

    Recent developments in optical systems (isotope-selective non-dispersive infrared spectrometry) for breath testing have provided a robust, low-cost option for undertaking (13)C analysis. Although these systems were initially developed for breath testing for Helicobacter pylori, they have an enormous potential as a soil science research tool. The relatively low cost of the equipment, US$15,000-25,000, is within the research budgets of most institutes or universities. The simplicity of the mechanisms and optical nature mean that the equipment requires relatively low maintenance and minimal training. Thus methods were developed to prepare soil and plant materials for analysis using the breath test analyser. Results that compare conventional mass spectrometric methods with the breath test analyser will be presented. In combination with simple (13)C-plant-labeling techniques it is possible to devise methods for estimating carbon sequestration under different agronomic management practices within a short time frame. This enables assessment of the carbon credit value of a particular agronomic practice, which can in turn be used by policy makers for decision-making purposes. For global understanding of the effect of agricultural practices on the carbon cycle, data are required from a range of cropping systems and agro-ecological zones. The method and the approach described will enable collection of hard data within a reasonable time.

  15. 13C-labeled mixed triglyceride breath test (13C MTG-BT) in healthy children and children with cystic fibrosis (CF) under pancreatic enzyme replacement therapy (PERT): a pilot study.

    PubMed

    Herzog, Denise C; Delvin, Edgard E; Albert, Caroline; Marcotte, Jacques E; Pelletier, Véronique A; Seidman, Ernest G

    2008-12-01

    The MTG-BT estimates the hydrolysis of triacyl-glycerols by pancreatic lipase, and appears attractive for monitoring exogenous lipase requirements in patients with exocrine pancreatic insufficiency. To assess the test's discrimination capacity and repeatability, 9 CF patients with PERT and 10 healthy children underwent the (13)C-MTG-BT twice, at a 2- to 4-week interval. The test distinguished well between patients with severe exocrine pancreatic insufficiency (SEPI) and healthy subjects. However, within-subject variability for postprandial per thousand(13)C-enrichment and postprandial % dose recovery (PDR) was high in both groups. Therefore, the (13)C-MTG-BT seems useful to distinguish between SEPI and normal exocrine pancreatic function, but requires further development to improve its repeatability.

  16. Use of 13C Labeled Carbon Tetrachloride to Demonstrate the Transformation to Carbon Dioxide under Anaerobic Conditions in a Continuous Flow Column

    NASA Astrophysics Data System (ADS)

    Semprini, L.; Azizian, M.

    2012-12-01

    The demonstration of transformation of chlorinated aliphatic compounds (CAHs) in the subsurface is a challenge, especially when the products are carbon dioxide (CO2) and chloride ion. The groundwater contaminant carbon tetrachloride (CT) is of particular interest since a broad range of transformation products can be potentially formed under anaerobic conditions. The ability to demonstrate the transformation of CT to CO2 as a non toxic endproduct, is also of great interest. Results will be presented from a continuous flow column study where 13C labeled CT was used to demonstrate its transformation to CO2. The column was packed with a quartz sand and bioaugmented the Evanite Culture (EV) that is capable of transforming tetrachloroethene (PCE) to ethene. The column was continously fed a synthetic groundwater that was amended with PCE (0.10 mM) and either formate (1.5 mM) or lactate (1.1 mM), which ferments to produce hydrogen (H2) as the ultimate electron donor. Earlier CT transformation studies with the column, in the absence of sulfate reduction, and with formate added as a donor found CT (0.015 mM) was over 98% transformed with about 20% converted to chloroform (CF) (0.003 mM) and with a transient detection of chloromethane (CM). Methane and carbon disulfide, as potential products, were not detected. Neither CT nor CF inhibited the reductive dehalogenation of PCE to ethene. A series of transient studies conducted after these initial CT transformation tests, but in the absence of CT, showed formate remained an effective substrate for maintaining sulfate reduction and PCE transformation. Lactate, which was effectively fermented prior to CT addition, was not effectively fermented, with propionate accumulating as a fermentation product. When lactate was added, PCE was mainly transformed to cis-dichloroethene (cis-DCE) and VC, and sulfate reduction did not occur. In order to restore effective lactate fermentation the column was then bioaugmented with an EV culture that

  17. Synthesis of Isotopically Labeled (13)C3-Simazine and Development of a Simultaneous UPLC-MS/MS Method for the Analysis of Simazine in Soil.

    PubMed

    Song, Yan; Guo, Yangzhen; Zhang, Xia; Yang, Yue; Chen, Shuo; She, Gaimei; She, Dongmei

    2016-01-14

    The isotope dilution mass spectrometry (IDMS) is a highly efficient method for tackling the ion suppression in complex matrix by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), but a lack of commercial internal standards is a limiting factor for these analyses. Herein, an economical and efficient strategy for the synthesis of (13)C3-simazine via a three-step procedure was developed. The isotope-labeled internal standard was used for determination of simazine residue in soil samples. The quantitation method has a limit of detection of 0.015 μg/kg and quantitation of 0.08 μg/kg. The inter-day and intra-day precision of the method were below 4.6%. Recovery values were ranged between 92.9% and 99.2%. All the samples obtained from six provinces in China contained from 1 to 62 μg/kg of simazine.

  18. [13C]-Specific labeling of 8-2' linked (-)-cis-blechnic, (-)-trans-blechnic and (-)-brainic acids in the fern Blechnum spicant

    NASA Technical Reports Server (NTRS)

    Davin, Laurence B.; Wang, Chang-Zeng; Helms, Gregory L.; Lewis, Norman G.

    2003-01-01

    In vivo administration experiments using stable (13C) and radio (14C) labeled precursors established that the optically active 8-2' linked lignans, (-)-cis-blechnic, (-)-trans-blechnic and (-)-trans-brainic acids, were directly derived from L-phenylalanine, cinnamate, and p-coumarate but not either from tyrosine or acetate. The radiochemical time course data suggest that the initial coupling product is (-)-cis-blechnic acid, which is then apparently converted into both (-)-trans-blechnic and (-)-trans-brainic acids in vivo. These findings provide additional evidence for vascular plant proteins engendering distinct but specific phenolic radical-radical coupling modes, i.e., for full control over phenylpropanoid coupling in vivo, whether stereoselective or regiospecific.

  19. Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry.

    PubMed

    Hart, David J; Finglas, Paul M; Wolfe, Caroline A; Mellon, Fred; Wright, Anthony J A; Southon, Susan

    2002-06-15

    The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.

  20. Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry.

    PubMed

    Hart, David J; Finglas, Paul M; Wolfe, Caroline A; Mellon, Fred; Wright, Anthony J A; Southon, Susan

    2002-06-15

    The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism. PMID:12054449

  1. More than a century of Grain for Green Program is expected to restore soil carbon stock on alpine grassland revealed by field (13)C pulse labeling.

    PubMed

    Li, Qi; Chen, Dongdong; Zhao, Liang; Yang, Xue; Xu, Shixiao; Zhao, Xinquan

    2016-04-15

    Anthropogenic changes in land use/cover have altered the vegetation, soil, and carbon (C) cycling on the Qinghai-Tibetan Plateau (QTP) over the last ~50years. As a result, the Grain for Green Program (GfGP) has been widely implemented over the last 10years to mitigate the impacts of cultivation. To quantify the effects of the GfGP on C partitioning and turnover rates at the ecosystem scale, an in situ (13)C pulse labeling experiment was conducted on natural and GfGP grasslands in an agro-pastoral ecotone in the Lake Qinghai region on the QTP. We found that there were significant differences in the C stocks of all the considered pools in both the natural and GfGP grasslands, with higher CO2 uptake rates in the GfGP grassland than that in the natural grassland. Partitioning of photoassimilate (% of recovered (13)C) in C pools of both grasslands was similar 25days after labeling, except in the roots of the 0-15 and 5-15cm soil layer. Soil organic C (SOC) sequestration rate in the GfGP grassland was 11.59±1.89gCm(-2)yr(-1) significantly greater than that in the natural grassland. The results confirmed that the GfGP is an efficient approach for grassland restoration and C sequestration. However, it will take more than a century (119.19±20.26yr) to restore the SOC stock from the current cropland baseline level to the approximate level of natural grassland. We suggest that additional measures are needed in the selection of suitable plant species for vegetation restoration, and in reasonable grazing management.

  2. The 2D {31P} Spin-Echo-Difference Constant-Time [13C, 1H]-HMQC Experiment for Simultaneous Determination of 3JH3‧P and 3JC4‧P in 13C-Labeled Nucleic Acids and Their Protein Complexes

    NASA Astrophysics Data System (ADS)

    Szyperski, Thomas; Fernández, César; Ono, Akira; Wüthrich, Kurt; Kainosho, Masatsune

    1999-10-01

    A two-dimensional {31P} spin-echo-difference constant-time [13C, 1H]-HMQC experiment (2D {31P}-sedct-[13C, 1H]-HMQC) is introduced for measurements of 3JC4‧P and 3JH3‧P scalar couplings in large 13C-labeled nucleic acids and in DNA-protein complexes. This experiment makes use of the fact that 1H-13C multiple-quantum coherences in macromolecules relax more slowly than the corresponding 13C single-quantum coherences. 3JC4‧P and 3JH3‧P are related via Karplus-type functions with the phosphodiester torsion angles β and ɛ, respectively, and their experimental assessment therefore contributes to further improved quality of NMR solution structures. Data are presented for a uniformly 13C, 15N-labeled 14-base-pair DNA duplex, both free in solution and in a 17-kDa protein-DNA complex.

  3. Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by 13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13C-dansyl and 12C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

  4. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  5. Laccase-catalyzed reactions of 17β-estradiol in the presence of humic acid: Resolved by high-resolution mass spectrometry in combination with (13)C labeling.

    PubMed

    Sun, Kai; Luo, Qi; Gao, Yanzheng; Huang, Qingguo

    2016-02-01

    The widespread presence of estrogens in natural waters poses potential threats to the aquatic organisms and human health. It is known that estrogens undergo enzyme-catalyzed oxidative coupling (ECOC) reactions, which may impact their environmental fate and can be used in wastewater treatment to remove estrogens, but little information is available on how natural organic matter (NOM) may influence 17β-estradiol (E2) transformation in ECOC processes. A series of experiments were conducted to examine the transformation of E2 in aqueous solution containing humic acid (HA) as model NOM by laccase-mediated ECOC reactions. The impact of HA on the reaction behaviors and product distribution is systematically characterized. The presence of HA inhibited the extent of E2 self-coupling in laccase-mediated systems, while promoted cross-coupling between E2 and HA. Reconfiguration of humic molecules was also observed and characterized by changes in absorbance at 275 nm and the ratios between A250 nm/A365 nm. In particular, experiments were conducted with un-labeled E2 mixed with (13)C3-labeled E2 at a set ratio, with the products probed using high-resolution mass spectrometry (HRMS). The high m/z accuracy of HRMS enabled the use of isotope ratio as a tracer to identify possible cross-coupling products between E2 and HA. Such a method combining HRMS and isotope labeling provides a novel means for identification of products in a reaction system involving NOM or other complex matrices. These findings provide a basis for optimization of ECOC reactions for estrogen removal, and also help to understand the environmental transformation of estrogens. PMID:26692517

  6. Priming effect of (13)C-labelled wheat straw in no-tillage soil under drying and wetting cycles in the Loess Plateau of China.

    PubMed

    Liu, Enke; Wang, Jianbo; Zhang, Yanqing; Angers, Denis A; Yan, Changrong; Oweis, Theib; He, Wenqing; Liu, Qin; Chen, Baoqing

    2015-09-08

    The objectives of this study were to determine the effects of drying and wetting (DW) cycles on soil organic carbon (SOC) mineralisation and on the priming effect (PE) induced by the addition of (13)C-labelled wheat straw to long-term no-tillage (NT) and conventional-tillage (CT) soils. We observed that the SOC mineralisation rate in rewetted soils was greater than that in soils that were kept at constant water content. The proportion of CO2 derived from the straw declined dramatically during the first 10 days. The priming direction was first positive, and then became slightly negative. The PE was higher under DW cycles than under constant water content. There was no significant effect of the tillage system on the SOC mineralisation rate or PE. The data indicate that the DW cycles had a significant effect on the SOC mineralisation rate and on the PE, demonstrating a positive combined effect between wheat straw and moisture fluctuations. Further research is needed to study the role of microbial communities and C pools in affecting the SOC mineralisation response to DW cycles.

  7. 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

  8. 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.

  9. Priming effect of 13C-labelled wheat straw in no-tillage soil under drying and wetting cycles in the Loess Plateau of China

    PubMed Central

    Liu, Enke; Wang, Jianbo; Zhang, Yanqing; Angers, Denis A.; Yan, Changrong; Oweis, Theib; He, Wenqing; Liu, Qin; Chen, Baoqing

    2015-01-01

    The objectives of this study were to determine the effects of drying and wetting (DW) cycles on soil organic carbon (SOC) mineralisation and on the priming effect (PE) induced by the addition of 13C-labelled wheat straw to long-term no-tillage (NT) and conventional-tillage (CT) soils. We observed that the SOC mineralisation rate in rewetted soils was greater than that in soils that were kept at constant water content. The proportion of CO2 derived from the straw declined dramatically during the first 10 days. The priming direction was first positive, and then became slightly negative. The PE was higher under DW cycles than under constant water content. There was no significant effect of the tillage system on the SOC mineralisation rate or PE. The data indicate that the DW cycles had a significant effect on the SOC mineralisation rate and on the PE, demonstrating a positive combined effect between wheat straw and moisture fluctuations. Further research is needed to study the role of microbial communities and C pools in affecting the SOC mineralisation response to DW cycles. PMID:26345303

  10. Orally Ingested 13C2-Retinol is Incorporated into Hepatic Retinyl Esters in a Nonhuman Primate (Macaca mulatta) Model of Hypervitaminosis A

    PubMed Central

    Escaron, Anne L; Tanumihardjo, Sherry A

    2010-01-01

    The mechanism responsible for the metabolism of vitamin A during hypervitaminosis is largely unknown. This study investigated hepatic 13C-retinol uptake in hypervitaminotic A rhesus monkeys. We hypothesized that individual retinyl esters would be enriched in 13C after a physiologic dose of 13C2-retinyl acetate, thus suggesting de novo in vivo hepatic retinol esterification. Male rhesus macaques (n = 16; 11.8 ± 2.9 y) each received 3.5 µmol 14, 15-13C2-retinyl acetate. Blood was drawn at baseline and 5 h and 2, 4, 7, 14, 21, and 28 d after administration. Liver biopsies were collected 7 d before and 2 d after dose administration (n = 4) and at 7, 14, and 28 d after dose administration (n = 4 per time point). 13C enrichments of retinol and retinyl esters HPLC-purified from liver samples were measured by using gas chromatography–combustion–isotope ratio mass spectrometry. 13C enrichment of total vitamin A and individual retinyl esters were significantly greater 2 d after dose administration compared with baseline levels. In contrast, the concentration of isolated retinyl esters did not always increase 2 d after treatment. Given that the liver biopsy site differed between monkeys, these data suggest that the accumulation of hepatic retinyl esters is a dynamic process that is better represented by combining analytical techniques. This sensitive methodology can be used to characterize vitamin A trafficking after physiologic doses of 13C-retinol. In this nonhuman primate model of hypervitaminosis A, hepatic retinyl esters continued to accumulate with high liver stores. PMID:20158952

  11. Direct incorporation of fatty acids into microbial phospholipids in soils: Position-specific labeling tells the story

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela A.; Kuzyakov, Yakov

    2016-02-01

    Fatty acids have been used as plant and microbial biomarkers, and knowledge about their transformation pathways in soils and sediments is crucial for interpreting fatty acid signatures, especially because the formation, recycling and decomposition processes are concurrent. We analyzed the incorporation of free fatty acids into microbial fatty acids in soil by coupling position-specific 13C labeling with compound-specific 13C analysis. Position-specifically and uniformly 13C labeled palmitate were applied in an agricultural Luvisol. Pathways of fatty acids were traced by analyzing microbial utilization of 13C from individual molecule positions of palmitate and their incorporation into phospholipid fatty acids (PLFA). The fate of palmitate 13C in the soil was characterized by the main pathways of microbial fatty acid metabolism: Odd positions (C-1) were preferentially oxidized to CO2 in the citric acid cycle, whereas even positions (C-2) were preferentially incorporated into microbial biomass. This pattern is a result of palmitate cleavage to acetyl-CoA and its further use in the main pathways of C metabolism. We observed a direct, intact incorporation of more than 4% of the added palmitate into the PLFA of microbial cell membranes, indicating the important role of palmitate as direct precursor for microbial fatty acids. Palmitate 13C was incorporated into PLFA as intact alkyl chain, i.e. the C backbone of palmitate was not cleaved, but palmitate was incorporated either intact or modified (e.g. desaturated, elongated or branched) according to the fatty acid demand of the microbial community. These modifications of the incorporated palmitate increased with time. Future PLFA studies must therefore consider the recycling of existing plant and microbial-derived fatty acids. This study demonstrates the intact uptake and recycling of free fatty acids such as palmitate in soils, as well as the high turnover and transformation of cellular PLFA. Knowledge about the intact

  12. 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+).

  13. The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

    PubMed Central

    2015-01-01

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

  14. Production process monitoring by serial mapping of microbial carbon flux distributions using a novel Sensor Reactor approach: II--(13)C-labeling-based metabolic flux analysis and L-lysine production.

    PubMed

    Drysch, A; El Massaoudi, M; Mack, C; Takors, R; de Graaf, A A; Sahm, H

    2003-04-01

    Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine. However, metabolic flux analyses based on 13C-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations. To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (El Massaoudi et al., Metab. Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling 13C-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail. We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of 13C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, 13C-based metabolic flux analysis was performed on exponentially growing, lysine-producing C. glutamicum MH20-22B during three phases of a pilot-scale batch fermentation. By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified. Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid 13C-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth. It was shown for the first time that the in vivo reverse C(4)-decarboxylation flux at the anaplerotic node in C. glutamicum significantly decreased (70%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth.

  15. Production of Hydrolysable Tannin-Like Structures During the Microbial Demethylation of lignin: An Assessment Using13C-Labeled Tetramethylammonium Hydroxide Thermochemolysis.

    NASA Astrophysics Data System (ADS)

    Filley, T.; Blanchette, R.; Nierop, K.; Gamblin, D.

    2003-12-01

    Phenolic compounds in soils are important mediators of microbial activity, metal mobility, soil redox, and soil organic matter building processes. Direct tannin input and the microbial decomposition of lignin in litter and soil are important contributors to this pool of phenols. The ability to accurately assess the relative differences in lignin decay (which are initiated by demethylation and side chain oxidation) among synapyl, coniferyl, and p-coumaryl components of detrital lignin requires the ability to determine microbial demethylation within the complex soil residues. Differentiating between hydrolysable tannins and contributions from advanced lignin decay can be problematic for many of the most common molecular techniques such as alkaline CuO oxidation, pyrolysis GC, and tetramethylammonium hydroxide thermochemolysis because of either the masking effects of derivatizing agents, oxidative damage to ortho-phenols or low volatility of lignin monomers. In this study we investigate lignin demethylation and polyhydroxyl-aromatic production in BC and C horizons of sandy forest soils dominated by oak, the A horizon from a red spruce forest, and controlled microbial inoculation studies of woody tissue using in-line 13C-labeled tetramethylammonium hydroxide thermochemolysis. Both white-rot and brown-rot decay resulted in syringyl demethylation, with the latter exhibiting more aggressive demethylation chemistry, while coniferyl monomer demethylation was essentially restricted to brown-rot decay. In a typical brown-rot sequence demethylation of syringyl components occurs more rapidly than coniferyl units within the same tissue and lower molecular weight fragments are likewise more demethylated than lignin monomers containing the full glycerol side chain. Demethylation of both methoxyl groups in the syringyl monomer is evident in soil horizons as well as laboratory inoculations. The latter may suggest demethylation after lignin depolymerization. Low molecular weight

  16. Refined Analysis of Brain Energy Metabolism Using In Vivo Dynamic Enrichment of 13C Multiplets

    PubMed Central

    Dehghani M., Masoumeh; Duarte, João M. N.; Kunz, Nicolas; Gruetter, Rolf

    2016-01-01

    Carbon-13 nuclear magnetic resonance spectroscopy in combination with the infusion of 13C-labeled precursors is a unique approach to study in vivo brain energy metabolism. Incorporating the maximum information available from in vivo localized 13C spectra is of importance to get broader knowledge on cerebral metabolic pathways. Metabolic rates can be quantitatively determined from the rate of 13C incorporation into amino acid neurotransmitters such as glutamate and glutamine using suitable mathematical models. The time course of multiplets arising from 13C-13C coupling between adjacent carbon atoms was expected to provide additional information for metabolic modeling leading to potential improvements in the estimation of metabolic parameters. The aim of the present study was to extend two-compartment neuronal/glial modeling to include dynamics of 13C isotopomers available from fine structure multiplets in 13C spectra of glutamate and glutamine measured in vivo in rats brain at 14.1 T, termed bonded cumomer approach. Incorporating the labeling time courses of 13C multiplets of glutamate and glutamine resulted in elevated precision of the estimated fluxes in rat brain as well as reduced correlations between them. PMID:26969691

  17. Aromatic spectral editing techniques for magic-angle-spinning solid-state NMR spectroscopy of uniformly (13)C-labeled proteins.

    PubMed

    Williams, Jonathan K; Schmidt-Rohr, Klaus; Hong, Mei

    2015-11-01

    The four aromatic amino acids in proteins, namely histidine, phenylalanine, tyrosine, and tryptophan, have strongly overlapping (13)C chemical shift ranges between 100 and 160ppm, and have so far been largely neglected in solid-state NMR determination of protein structures. Yet aromatic residues play important roles in biology through π-π and cation-π interactions. To better resolve and assign aromatic residues' (13)C signals in magic-angle-spinning (MAS) solid-state NMR spectra, we introduce two spectral editing techniques. The first method uses gated (1)H decoupling in a proton-driven spin-diffusion (PDSD) experiment to remove all protonated (13)C signals and retain only non-protonated carbon signals in the aromatic region of the (13)C spectra. The second technique uses chemical shift filters and (1)H-(13)C dipolar dephasing to selectively detect the Cα, Cβ and CO cross peaks of aromatic residues while suppressing the signals of all aliphatic residues. We demonstrate these two techniques on amino acids, a model peptide, and the microcrystalline protein GB1, and show that they significantly simplify the 2D NMR spectra and both reveal and permit the ready assignment of the aromatic residues' signals.

  18. Transformation of 17β-estradiol in humic acid solution by ε-MnO2 nanorods as probed by high-resolution mass spectrometry combined with (13)C labeling.

    PubMed

    Sun, Kai; Liang, Shangtao; Kang, Fuxing; Gao, Yanzheng; Huang, Qingguo

    2016-07-01

    Steroidal estrogens (SEs), widespread in aquatic systems, have a potential to disrupt the endocrine system of wildlife species and humans. In our experiments, the performance of ε-MnO2 nanorods in transforming 17β-estradiol (E2) was investigated, and the effect of humic acid (HA) on the reaction behaviors was systematically characterized. Reconfiguration of humic molecules was also investigated by high-performance size exclusion chromatography (HPSEC). Results indicated that ε-MnO2 nanomaterials ensured efficient removal of E2 from the aqueous solution. The presence of HA hindered the transformation of E2, while enhanced the cross-coupling between E2 and humic molecules. In particular, we used a mixture of un-labeled E2 and (13)C3-labeled E2 at a 1: 1 set ratio (w/w) to probe the reaction products via high-resolution mass spectrometry (HRMS). The combination of HRMS and (13)C3-labeling revealed the intermediate products including estrone (E1), and hydroxylated, quinone-like, and ring-opened species, as well as E2 dimer and trimer. More importantly, possible cross-coupling products between E2 and HA were also identified. A reaction mechanism including two-electron oxidation and single-electron oxidation was proposed. The applied analytical approach using HRMS along with (13)C3-labeling for reaction-product identification is crucial to understanding the role of HA in the transformation of SEs.

  19. Transformation of 17β-estradiol in humic acid solution by ε-MnO2 nanorods as probed by high-resolution mass spectrometry combined with (13)C labeling.

    PubMed

    Sun, Kai; Liang, Shangtao; Kang, Fuxing; Gao, Yanzheng; Huang, Qingguo

    2016-07-01

    Steroidal estrogens (SEs), widespread in aquatic systems, have a potential to disrupt the endocrine system of wildlife species and humans. In our experiments, the performance of ε-MnO2 nanorods in transforming 17β-estradiol (E2) was investigated, and the effect of humic acid (HA) on the reaction behaviors was systematically characterized. Reconfiguration of humic molecules was also investigated by high-performance size exclusion chromatography (HPSEC). Results indicated that ε-MnO2 nanomaterials ensured efficient removal of E2 from the aqueous solution. The presence of HA hindered the transformation of E2, while enhanced the cross-coupling between E2 and humic molecules. In particular, we used a mixture of un-labeled E2 and (13)C3-labeled E2 at a 1: 1 set ratio (w/w) to probe the reaction products via high-resolution mass spectrometry (HRMS). The combination of HRMS and (13)C3-labeling revealed the intermediate products including estrone (E1), and hydroxylated, quinone-like, and ring-opened species, as well as E2 dimer and trimer. More importantly, possible cross-coupling products between E2 and HA were also identified. A reaction mechanism including two-electron oxidation and single-electron oxidation was proposed. The applied analytical approach using HRMS along with (13)C3-labeling for reaction-product identification is crucial to understanding the role of HA in the transformation of SEs. PMID:27086077

  20. Insights into the metabolic response to traumatic brain injury as revealed by 13C NMR spectroscopy

    PubMed Central

    Bartnik-Olson, Brenda L.; Harris, Neil G.; Shijo, Katsunori; Sutton, Richard L.

    2013-01-01

    The present review highlights critical issues related to cerebral metabolism following traumatic brain injury (TBI) and the use of 13C labeled substrates and nuclear magnetic resonance (NMR) spectroscopy to study these changes. First we address some pathophysiologic factors contributing to metabolic dysfunction following TBI. We then examine how 13C NMR spectroscopy strategies have been used to investigate energy metabolism, neurotransmission, the intracellular redox state, and neuroglial compartmentation following injury. 13C NMR spectroscopy studies of brain extracts from animal models of TBI have revealed enhanced glycolytic production of lactate, evidence of pentose phosphate pathway (PPP) activation, and alterations in neuronal and astrocyte oxidative metabolism that are dependent on injury severity. Differential incorporation of label into glutamate and glutamine from 13C labeled glucose or acetate also suggest TBI-induced adaptations to the glutamate-glutamine cycle. PMID:24109452

  1. 13C-1H dipolar-driven 13C-13C recoupling without 13C rf irradiation in nuclear magnetic resonance of rotating solids

    NASA Astrophysics Data System (ADS)

    Takegoshi, K.; Nakamura, Shinji; Terao, Takehiko

    2003-02-01

    the spinning speed. Further, we showed that the efficiency of the second-order DARR recoupling is not significantly less than that of the first-order DARR. Among the 13C-1H recoupling methods examined, CW irradiation at the n=1 rotary-resonance condition is superior for DARR because it gives a larger 13C-1H dipolar broadening, leading to broadband recoupling. We showed that a broadband-recoupling experiment with the first and the second-order DARR by CW irradiation at the n=1 rotary-resonance condition is applicable to signal assignment as well as structural determination of a multiply/uniformly 13C labeled molecule as demonstrated by two-dimensional 13C-13C DARR polarization-transfer experiments of uniformly 13C, 15N-labeled glycylisoleucine.

  2. Metabolic pathways for ketone body production. /sup 13/C NMR spectroscopy of rat liver in vivo using /sup 13/C-multilabeled fatty acids

    SciTech Connect

    Pahl-Wostl, C.; Seelig, J.

    1986-11-04

    The hormonal regulation of ketogenesis in the liver of living rat has been studied noninvasively with /sup 13/C nuclear magnetic resonance. The spatial selection for the liver was better than 90%, with extrahepatic adipose tissue contribution only a very small amount of signal. The metabolic activities of the liver were investigated by infusion of /sup 13/C-labeled butyrate in the jugular vein of the anesthetized rat. The rate of butyrate infusion was chosen to be close to the maximum oxidative capacity of the rat liver, and the /sup 13/C signal intensities were enhanced by using doubly labeled (1,3-/sup 13/C)butyrate as a substrate. Different /sup 13/C NMR spectra and hence different metabolites were observed depending on the hormonal state of the animal. The /sup 13/C NMR studies demonstrate that even when rate of acetyl-CoA production are high, the disposal of this compound is not identical in fasted and diabetic animals. This supports previous suggestions that the redox state of the mitochondrion represents the most important factor in regulation. For a given metabolic state of the animal, different signal intensities were obtained depending on whether butyrate was labeled at C-1, C-3, or C-1,3. From the ratios of incorporation of /sup 13/C label into the carbons of 3-hydroxybutyrate, it could be estimated that a large fraction of butyrate evaded ..beta..-oxidation to acetyl-CoA but was converted directly to acetoacetyl-CoA. /sup 13/C-labeled glucose could be detected in vivo in the liver of diabetic rats.

  3. Synthesis Of 2h- And 13c-Substituted Dithanes

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.

    2004-05-04

    The present invention is directed to labeled compounds, [2-.sup.13 C]dithane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to processes of preparing [2-.sup.13 C]dithane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to labeled compounds, e.g., [.sup.2 H.sub.1-2, .sup.13 C]methanol (arylthio)-, acetates wherein the .sup.13 C atom is directly bonded to exactly one or two deuterium atoms.

  4. Synthesis of 2H- and 13C-substituted dithanes

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.

    2003-01-01

    The present invention is directed to labeled compounds, [2-.sup.13 C]dithiane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to processes of preparing [2-.sup.13 C]dithiane wherein the .sup.13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to labeled compounds, e.g., [.sup.2 H.sub.1-2, .sup.13 C]methanol (arylthio)-, acetates wherein the .sup.13 C atom is directly bonded to exactly one or two deuterium atoms.

  5. Folate is absorbed across the human colon: evidence by using enteric-coated caplets containing 13C-labeled [6S]-5-formyltetrahydrofolate1, 2, 3, 4

    PubMed Central

    Lakoff, Alanna; Fazili, Zia; Aufreiter, Susanne; Pfeiffer, Christine M; Connolly, Bairbie; Gregory, Jesse F; Pencharz, Paul B; O’Connor, Deborah L

    2016-01-01

    Background Folate intakes that do not meet or greatly exceed requirements may be associated with negative health outcomes. A better understanding of contributors that influence the input side will help establish dietary guidance that ensures health benefits without associated risks. Colonic microbiota produce large quantities of folate, and [13C5]5-formyltetrahydrofolate infused during colonoscopy is absorbed. However, it is unclear if significant quantities of folate are absorbed in an intact microbiome. Objective We determined whether and how much of a physiologic dose of [13C5]5-formyltetrahydrofolate delivered in a pH-sensitive enteric caplet to an intact colonic microbiome is absorbed. Design Healthy adults ingested a specially designed pH-sensitive acrylic copolymer–coated barium sulfate caplet that contained 855 nmol (400 μg) [13C5]5-formyltetrahydrofolate. After a washout period ≥4 wk, subjects received an intravenous injection of the same compound (214 nmol). Serially collected blood samples before and after each test dose were analyzed by using a microbiological assay and liquid chromatography–tandem mass spectrometry. Results Caplet disintegration in the colon was observed by fluoroscopic imaging for 6 subjects with a mean (±SD) complete disintegration time of 284 ± 155 min. The mean (±SEM) rate of appearance of [13C5]5-methyltetrahydrofolate in plasma was 0.33 ± 0.09 (caplet) and 5.8 ± 1.2 (intravenous) nmol/h. Likely because of the significant time in the colon, the mean apparent absorption across the colon was 46%. Conclusions Folate is absorbed across the colon in humans with an undisturbed microbiome. This finding and previous observations of the size of the colonic depot of folate and its potential for manipulation by diet (eg, dietary fiber, oligosaccharides, and probiotics) suggest that an individual’s dietary folate requirement may differ depending on the consumption of dietary constituents that affect the size and composition of

  6. Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association.

    PubMed

    Molero, Gemma; Aranjuelo, Iker; Teixidor, Pilar; Araus, José Luis; Nogués, Salvador

    2011-03-15

    We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.

  7. In Situ 13C and 23Na Magic Angle Spinning NMR Investigation of Supercritical CO2 Incorporation in Smectite-Natural Organic Matter Composites

    SciTech Connect

    Bowers, Geoffrey M.; Hoyt, David W.; Burton, Sarah D.; Ferguson, Brennan O.; Varga, Tamas; Kirkpatrick, Robert J.

    2014-01-29

    This paper presents an in situ NMR study of clay-natural organic polymer systems (a hectoritehumic acid [HA] composite) under CO2 storage reservoir conditions (90 bars CO2 pressure, 50°C). The 13C and 23Na NMR data show that supercritical CO2 interacts more strongly with the composite than with the base clay and does not react to form other C-containing species over several days at elevated CO2. With and without organic matter, the data suggest that CO2 enters the interlayer space of Na-hectorite equilibrated at 43% relative humidity. The presence of supercritical CO2 also leads to increased 23Na signal intensity, reduced line width at half height, increased basal width, more rapid 23Na T1 relaxation rates, and a shift to more positive resonance frequencies. Larger changes are observed for the hectorite-HA composite than for the base clay. In light of recently reported MD simulations of other polymer-Na-smectite composites, we interpret the observed changes as an increase in the rate of Na+ site hopping in the presence of supercritical CO2, the presence of potential new Na+ sorption sites when the humic acid is present, and perhaps an accompanying increase in the number of Na+ ions actively involved in site hopping. The results suggest that the presence of organic material either in clay interlayers or on external particle surfaces can significantly affect the behavior of supercritical CO2 and the mobility of metal ions in reservoir rocks.

  8. (13)C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging.

    PubMed

    Salamanca-Cardona, Lucia; Keshari, Kayvan R

    2015-01-01

    In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-(13)C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.

  9. 13 C solid-state NMR study of the 13 C-labeled peptide, (E)8 GGLGGQGAG(A)6 GGAGQGGYGG as a model for the local structure of Nephila clavipes dragline silk (MaSp1) before and after spinning.

    PubMed

    Yazawa, Koji; Yamaguchi, Erika; Knight, David; Asakura, Tetsuo

    2012-06-01

    We prepared the water soluble model peptide, (E)(8) GGLGGQGAG(A)(6) GGAGQGGYGG, to throw light on the local structure of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes before and after spinning. Solution (13) C NMR showed that the conformation of the peptide in aqueous solution was essentially random coil. Solid-state NMR was used to follow conformation-dependent (13) C chemical shifts in (13) C selectively labeled versions of the peptide. The peptide lyophilized from an aqueous solution at neutral pH (hereafter referred to as "without acid treatment)"was used to mimic the state of the spidroin stored in the spider's silk gland while the peptide precipitated from the acidic solution ("with acid treatment") was used to simulate the role of acid treatment in inducing conformation change in the natural spinning process. In without acid treatment, the fraction of random coil conformation was lowest in the N-terminal region (residues 15-18) when compared with the C-terminus. The conformational change produced by the acid treatment occurred in the sequence, G(15) AG(A)(6) GGAG(27), interposed between pairs of Gly residues pairs, Gly(12,13), and Gly(29,30). The acid treated peptide showed a remarkable decrease in the fraction of random coil conformation from A(20) to A(23) in the poly-Ala region when compared with the peptide without acid treatment. These observations taken together suggest that the peptide can be used as a model for studying the localization of the conformation change in spider silk fibroin in the natural spinning and the role of acid treatment in this process.

  10. In vivo hyperpolarized 13C MR spectroscopic imaging with 1H decoupling

    NASA Astrophysics Data System (ADS)

    Chen, Albert P.; Tropp, James; Hurd, Ralph E.; Van Criekinge, Mark; Carvajal, Lucas G.; Xu, Duan; Kurhanewicz, John; Vigneron, Daniel B.

    2009-03-01

    Application of 13C MRS in vivo on whole body MR system has been limited due to the low static field (and consequent low signal to noise ratio—SNR) of these scanners; thus there have been few reports of 1H decoupled 13C MRS in vivo using a clinical MR platform. The recent development of techniques to retain highly polarized spins in solution following DNP in a solid matrix has provided a mechanism to use endogenous pre-polarized 13C labeled substrates to study real time cellular metabolism in vivo with high SNR. In a recent in vivo hyperpolarized metabolic imaging study using 13C pyruvate, it has been demonstrated that the line shape (signal decay) of the resonances observed are greatly affected by JCH coupling in addition to inhomogeneous broadening. This study demonstrates the feasibility of improving hyperpolarized 13C metabolic imaging in vivo by incorporating 1H decoupling on a clinical whole body 3 T MR scanner. No reduction of T1 of a pre-polarized 13C substrate ([1- 13C] lactate) in solution was observed when 1H decoupling was applied with WALTZ16 sequence. Narrower linewidth for the [1- 13C] lactate resonance was observed in hyperpolarized 13C MRSI data in vivo with 1H decoupling.

  11. Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids

    NASA Astrophysics Data System (ADS)

    Potapov, Alexey; Yau, Wai-Ming; Tycko, Robert

    2013-06-01

    We explore the possibility of using dynamic nuclear polarization (DNP) to enhance signals in structural studies of biological solids by solid state NMR without sample spinning. Specifically, we use 2D 13C-13C exchange spectroscopy to probe the peptide backbone torsion angles (ϕ, ψ) in a series of selectively 13C-labeled 40-residue β-amyloid (Aβ1-40) samples, in both fibrillar and non-fibrillar states. Experiments are carried out at 9.39 T and 8 K, using a static double-resonance NMR probe and low-power microwave irradiation at 264 GHz. In frozen solutions of Aβ1-40 fibrils doped with DOTOPA-TEMPO, we observe DNP signal enhancement factors of 16-21. We show that the orientation- and frequency-dependent spin polarization exchange between sequential backbone carbonyl 13C labels can be simulated accurately using a simple expression for the exchange rate, after experimentally determined homogeneous 13C lineshapes are incorporated in the simulations. The experimental 2D 13C-13C exchange spectra place constraints on the ϕ and ψ angles between the two carbonyl labels. Although the data are not sufficient to determine ϕ and ψ uniquely, the data do provide non-trivial constraints that could be included in structure calculations. With DNP at low temperatures, 2D 13C-13C exchange spectra can be obtained from a 3.5 mg sample of Aβ1-40 fibrils in 4 h or less, despite the broad 13C chemical shift anisotropy line shapes that are observed in static samples.

  12. Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids

    PubMed Central

    Potapov, Alexey; Yau, Wai-Ming; Tycko, Robert

    2013-01-01

    We explore the possibility of using dynamic nuclear polarization (DNP) to enhance signals in structural studies of biological solids by solid state NMR without sample spinning. Specifically, we use 2D 13C-13C exchange spectroscopy to probe the peptide backbone torsion angles (ϕ,ψ) in a series of selectively 13C-labeled 40-residue β-amyloid (Aβ1–40) samples, in both fibrillar and non-fibrillar states. Experiments are carried out at 9.39 T and 8 K, using a static double-resonance NMR probe and low-power microwave irradiation at 264 GHz. In frozen solutions of Aβ1–40 fibrils doped with DOTOPA-TEMPO, we observe DNP signal enhancement factors of 16–21. We show that the orientation- and frequency-dependent spin polarization exchange between sequential backbone carbonyl 13C labels can be simulated accurately using a simple expression for the exchange rate, after experimentally determined homogeneous 13C lineshapes are incorporated in the simulations. The experimental 2D 13C-13C exchange spectra place constraints on the ϕ and ψ angles between the two carbonyl labels. Although the data are not sufficient to determine ϕ and ψ uniquely, the data do provide non-trivial constraints that could be included in structure calculations. With DNP at low temperatures, 2D 13C-13C exchange spectra can be obtained from a 3.5 mg sample of Aβ1–40 fibrils in 4 hr or less, despite the broad 13C chemical shift anisotropy line shapes that are observed in static samples. PMID:23562665

  13. 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

  14. Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by 13C NMR Spectroscopy

    PubMed Central

    Ashworth, Dennis J.; Lee, Rino Y.; Adams, Douglas O.

    1987-01-01

    Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-13C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-13C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the 13C NMR spectra due to loss of the label as 13CO2. Uptake of [3-13C]pyruvate by the cells was considerably slower than with [2-13C]acetate; however, the labelling patterns were similar with the exception of increased [3-13C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-13C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine. PMID:16665721

  15. Mass spectrometry-based microassay of (2)H and (13)C plasma glucose labeling to quantify liver metabolic fluxes in vivo.

    PubMed

    Hasenour, Clinton M; Wall, Martha L; Ridley, D Emerson; Hughey, Curtis C; James, Freyja D; Wasserman, David H; Young, Jamey D

    2015-07-15

    Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.

  16. 13C Tracking after 13CO2 Supply Revealed Diurnal Patterns of Wood Formation in Aspen.

    PubMed

    Mahboubi, Amir; Linden, Pernilla; Hedenström, Mattias; Moritz, Thomas; Niittylä, Totte

    2015-06-01

    Wood of trees is formed from carbon assimilated in the photosynthetic tissues. Determining the temporal dynamics of carbon assimilation, subsequent transport into developing wood, and incorporation to cell walls would further our understanding of wood formation in particular and tree growth in general. To investigate these questions, we designed a (13)CO2 labeling system to study carbon transport and incorporation to developing wood of hybrid aspen (Populus tremula × tremuloides). Tracking of (13)C incorporation to wood over a time course using nuclear magnetic resonance spectroscopy revealed diurnal patterns in wood cell wall biosynthesis. The dark period had a differential effect on (13)C incorporation to lignin and cell wall carbohydrates. No (13)C was incorporated into aromatic amino acids of cell wall proteins in the dark, suggesting that cell wall protein biosynthesis ceased during the night. The results show previously unrecognized temporal patterns in wood cell wall biosynthesis, suggest diurnal cycle as a possible cue in the regulation of carbon incorporation to wood, and establish a unique (13)C labeling method for the analysis of wood formation and secondary growth in trees.

  17. The Ratio of Acetate-to-Glucose Oxidation in Astrocytes from a Single 13C NMR Spectrum of Cerebral Cortex

    PubMed Central

    Marin-Valencia, Isaac; Hooshyar, M. Ali; Pichumani, Kumar; Sherry, A. Dean; Malloy, Craig R.

    2014-01-01

    The 13C labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of 13C-enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α-ketoglutarate into glutamate in neurons, and incorporation of α-ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single 13C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate to glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2-13C]acetate and [1,6-13C]glucose, and proton decoupled 13C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that 13C-labeled acetate and glucose contributed approximately equally to acetyl-CoA (0.96) in astrocytes. Since this method relies on a single 13C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions. PMID:25231025

  18. Measurement of 1J(Ni,Calpha(i)), 1J(Ni,C'i-1), 2J(Ni,Calpha(i-1)), 2J(H(N)i,C'i-1) and 2J(H(N)i,Calpha(i)) values in 13C/15N-labeled proteins.

    PubMed

    Mukherjee, Sulakshana; Mustafi, Sourajit M; Atreya, H S; Chary, K V R

    2005-04-01

    Use of partial or selective (13)C/(15)N labeling of specific amino acid residues in a given protein to measure the values of (1)J((15)N(i),(13)C(alpha) (i)), (2)J((1)H(N),(13)C(alpha) (i)), (2)J((15)N(i),(13)C(alpha) (i-1)), (1)J((15)N(i),(13)C'(i-1)) and (2)J((1)H(N),(13)C'(i-1)) is described. This was achieved by recording a sensitivity-enhanced 2D [(15)N-(1)H] HSQC experiment, without mixing the spin states of C(alpha) and C' during the course of entire experiment.

  19. 13C pulse-labeling assessment of the community structure of active fungi in the rhizosphere of a genetically starch-modified potato (Solanum tuberosum) cultivar and its parental isoline.

    PubMed

    Hannula, S E; Boschker, H T S; de Boer, W; van Veen, J A

    2012-05-01

    • The aim of this study was to gain understanding of the carbon flow from the roots of a genetically modified (GM) amylopectin-accumulating potato (Solanum tuberosum) cultivar and its parental isoline to the soil fungal community using stable isotope probing (SIP). • The microbes receiving (13)C from the plant were assessed through RNA/phospholipid fatty acid analysis with stable isotope probing (PLFA-SIP) at three time-points (1, 5 and 12 d after the start of labeling). The communities of Ascomycota, Basidiomycota and Glomeromycota were analysed separately with RT-qPCR and terminal restriction fragment length polymorphism (T-RFLP). • Ascomycetes and glomeromycetes received carbon from the plant as early as 1 and 5 d after labeling, while basidiomycetes were slower in accumulating the labeled carbon. The rate of carbon allocation in the GM variety differed from that in its parental variety, thereby affecting soil fungal communities. • We conclude that both saprotrophic and mycorrhizal fungi rapidly metabolize organic substrates flowing from the root into the rhizosphere, that there are large differences in utilization of root-derived compounds at a lower phylogenetic level within investigated fungal phyla, and that active communities in the rhizosphere differ between the GM plant and its parental cultivar through effects of differential carbon flow from the plant.

  20. Synthesis Of [2h, 13c] And [2h3, 13c]Methyl Aryl Sulfides

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.

    2004-03-30

    The present invention is directed to labeled compounds, [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2, .sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfides wherein the .sup.13 C methyl group attached to the sulfur of the sulfide includes exactly one, two or three deuterium atoms and the aryl group is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR1## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are each independently, hydrogen, a C.sub.1 -C.sub.4 lower alkyl, a halogen, an amino group from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each a C.sub.1 -C.sub.4 lower alkyl, a phenyl, or an alkoxy group. The present invention is also directed to processes of preparing [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2,.sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfides wherein the .sup.13 C methyl group attached to the sulfur of the sulfide includes exactly one, two or three deuterium atoms. The present invention is also directed to the labeled compounds of [.sup.2 H.sub.1, .sup.13 C]methyl iodide and [.sup.2 H.sub.2, .sup.13 C]methyl iodide.

  1. Detection of Reduced GABA Synthesis Following Inhibition of GABA Transaminase Using in Vivo Magnetic Resonance Signal of [13C]GABA C1

    PubMed Central

    Yang, Jehoon; Johnson, Christopher; Shen, Jun

    2009-01-01

    Previous in vivo magnetic resonance spectroscopy (MRS) studies of gamma-aminobutyric acid (GABA) synthesis have relied on 13C label incorporation into GABA C2 from [1-13C] or [1,6-13C2]glucose. In this study, the [13C]GABA C1 signal at 182.3 ppm in the carboxylic/amide spectral region of localized in vivo 13C spectra was detected. GABA-transaminase of rat brain was inhibited by administration of gabaculine after pre-labeling of GABA C1 and its metabolic precursors with exogenous [2,5-13C2]glucose. A subsequent isotope chase experiment was performed by infusing unlabeled glucose, which revealed a markedly slow change in the labeling of GABA C1 accompanying the blockade of the GABA shunt. This slow labeling of GABA at elevated GABA concentration was attributed to the relatively small intercompartmental GABA-glutamine cycling flux that constitutes the main route of 13C label loss during the isotope chase. Because this study showed that using low RF power broadband stochastic proton decoupling is feasible at very high field strength, it has important implications for the development of carboxylic/amide 13C MRS methods to study brain metabolism and neurotransmission in human subjects at high magnetic fields. PMID:19540876

  2. /sup 13/C nuclear magnetic resonance studies of cardiac metabolism

    SciTech Connect

    Seeholzer, S.H.

    1985-01-01

    The last decade has witnessed the increasing use of Nuclear Magnetic Resonance (NMR) techniques for following the metabolic fate of compounds specifically labeled with /sup 13/C. The goals of the present study are: (1) to develop reliable quantitative procedures for measuring the /sup 13/C enrichment of specific carbon sites in compounds enriched by the metabolism of /sup 13/C-labeled substrates in rat heart, and (2) to use these quantitative measurements of fractional /sup 13/C enrichment within the context of a mathematical flux model describing the carbon flow through the TCA cycle and ancillary pathways, as a means for obtaining unknown flux parameters. Rat hearts have been perfused in vitro with various combinations of glucose, acetate, pyruvate, and propionate to achieve steady state flux conditions, followed by perfusion with the same substrates labeled with /sup 13/C in specific carbon sites. The hearts were frozen at different times after addition of /sup 13/C-labeled substrates and neutralized perchloric acid extracts were used to obtain high resolution proton-decoupled /sup 13/C NMR spectra at 90.55 MHz. The fractional /sup 13/C enrichment (F.E.) of individual carbon sites in different metabolites was calculated from the area of the resolved resonances after correction for saturation and nuclear Overhauser effects. These F.E. measurements by /sup 13/C NMR were validated by the analysis of /sup 13/C-/sup 1/H scalar coupling patterns observed in /sup 1/H NMR spectra of the extracted metabolites. The results obtained from perfusion of hearts glucose plus either (2-/sup 13/C) acetate or (3-/sup 13/C) pyruvate are similar to those obtained by previous investigators using /sup 14/C-labeled substrates.

  3. Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17. Structure elucidation and NMR study of a 13C,15N-labelled gyrase inhibitor.

    PubMed

    Bayer, A; Freund, S; Jung, G

    1995-12-01

    Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post-translational modification of the 69-residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43-residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non-labelled, 15N-labelled and 13C/15N-labelled peptide. By ultraviolet-absorption spectroscopy. HPLC-electrospray mass spectrometry and GC-mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly-Cys and Gly-Ser segments leads to the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-aminomethyloxazole-4-carboxylic acid, respectively. In addition, two bicyclic modifications 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase-inhibiting activity, e.g. interacting with the DNA to trap the covalent protein-DNA intermediate of the breakage-reunion reaction of the gyrase.

  4. 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

  5. sup 13 C-enriched ribonucleosides: Synthesis and application of sup 13 C- sup 1 H and sup 13 C- sup 13 C spin-coupling constants to assess furanose and N-glycoside bond conformations

    SciTech Connect

    Kline, P.C.; Serianni, A.S. )

    1990-09-26

    Adenosine (1), cytidine (2), guanosine (3), and uridine (4) have been prepared chemically with {sup 13}C enrichment (99 atom %) at C1{prime} and C2{prime} of the ribose ring. Reliable synthetic protocols have been developed to permit access to millimole quantities of labeled ribonucleosides required for structural studies of stable isotopically labeled oligonucleotides and for in vivo metabolism studies. High-resolution {sup 1}H and {sup 13}C NMR spectra of the enriched ribonucleosides have been obtained, and {sup 13}C-{sup 13}C and {sup 13}C-{sup 1}H spin-coupling constants have been measured for pathways within the {beta}-D-ribofuranose ring and across the N-glycoside bond. Related couplings were determined in methyl {alpha}- and {beta}-D-riboruanosides (5,6), and in two conformationally constrained nucleosides, 2,2{prime}-anhydro-(1-{beta}-D-arabinofuranosyl)uracil (7) and 2{prime},3{prime}-O-isopropylidene-2,5{prime}-O-cyclouridine (8). The latter data were used to construct a crude Karplus curve for the {sup 13}C-C-N-{sup 13}C coupling pathway across the N-glycoside bond in 1-4. {sup 1}H-{sup 1}H, {sup 13}C-{sup 1}H, and {sup 13}C-{sup 13}C coupling data are used to evaluate current models describing the conformational dynamics of 1-4 in aqueous solution.

  6. Biomimetic oxidative treatment of spruce wood studied by pyrolysis-molecular beam mass spectrometry coupled with multivariate analysis and 13C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood.

    PubMed

    Arantes, Valdeir; Qian, Yuhui; Kelley, Stephen S; Milagres, Adriane M F; Filley, Timothy R; Jellison, Jody; Goodell, Barry

    2009-11-01

    In this work, pyrolysis-molecular beam mass spectrometry analysis coupled with principal components analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis were used to study lignin oxidation, depolymerization, and demethylation of spruce wood treated by biomimetic oxidative systems. Neat Fenton and chelator-mediated Fenton reaction (CMFR) systems as well as cellulosic enzyme treatments were used to mimic the nonenzymatic process involved in wood brown-rot biodegradation. The results suggest that compared with enzymatic processes, Fenton-based treatment more readily opens the structure of the lignocellulosic matrix, freeing cellulose fibrils from the matrix. The results demonstrate that, under the current treatment conditions, Fenton and CMFR treatment cause limited demethoxylation of lignin in the insoluble wood residue. However, analysis of a water-extractable fraction revealed considerable soluble lignin residue structures that had undergone side chain oxidation as well as demethoxylation upon CMFR treatment. This research has implications for our understanding of nonenzymatic degradation of wood and the diffusion of CMFR agents in the wood cell wall during fungal degradation processes.

  7. Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products.

    PubMed

    Lohrey, Lilia; Marschik, Stefanie; Cramer, Benedikt; Humpf, Hans-Ulrich

    2013-01-01

    Tenuazonic acid is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. This paper describes a new strategy for the efficient and economical large-scale synthesis of the isotopically labeled internal standard (13)C(2)-tenuazonic acid via a three-step procedure. Furthermore, a new reliable and quick method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) cleanup is presented for the determination of tenuazonic acid in food and feed samples utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by application of the stable isotope dilution analysis. This new method has a limit of detection (LOD) of 0.86 μg/kg and a limit of quantitation (LOQ) of 2.89 μg/kg. In total 26 tomato samples and 4 bell pepper samples from the German market were analyzed. Tenuazonic acid was found in each sample with levels from 3 to 2330 μg/kg.

  8. 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.

  9. Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products.

    PubMed

    Lohrey, Lilia; Marschik, Stefanie; Cramer, Benedikt; Humpf, Hans-Ulrich

    2013-01-01

    Tenuazonic acid is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. This paper describes a new strategy for the efficient and economical large-scale synthesis of the isotopically labeled internal standard (13)C(2)-tenuazonic acid via a three-step procedure. Furthermore, a new reliable and quick method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) cleanup is presented for the determination of tenuazonic acid in food and feed samples utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by application of the stable isotope dilution analysis. This new method has a limit of detection (LOD) of 0.86 μg/kg and a limit of quantitation (LOQ) of 2.89 μg/kg. In total 26 tomato samples and 4 bell pepper samples from the German market were analyzed. Tenuazonic acid was found in each sample with levels from 3 to 2330 μg/kg. PMID:23230907

  10. Uptake and incorporation of labeled tryptophan isomers into IAA in the jsR sub 1 mutant of Lemna gibba

    SciTech Connect

    Baldi, B.G.; Maher, B.R.; Cohen, J.D. )

    1989-04-01

    Analyses of the IAA-overproducing mutant of Lemna have been initiated in order to study in vivo biosynthesis of IAA. Using radiolabelled tryptophan isomers prepared from commercial sources of {sup 14}C-D,L tryptophan by chiral separation kinetics of uptake of L and D tryptophan were determined for sterile cultures of individual jsR{sub 1} four-frond colonies. Over a 24 h period, about 50% of the radioactivity from {sup 14}C-L-TRP in media, or about 25% from {sup 14}C-D-TRP, was found in the plant tissue. Maximal rates of uptake were seen in the first six hors for both isomers. Endogenous levels of tryptophan determined in jsR{sub 1} as measures of pool sizes in vivo show 5 to 10 ug/g FW total tryptophan with less than 1% in the D isomer form. Information on uptake and endogenous pool sizes of tryptophan isomers is being used for feeding of stable isotope labeled tryptophan ({sup 13}C, {sup 14}N) to jsR{sub 1} at physiological levels. Analyses of incorporation of label into IAA using GC-MS and high resolution mass spectrometry are currently underway.

  11. Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

    PubMed

    Rabe, Patrick; Barra, Lena; Rinkel, Jan; Riclea, Ramona; Citron, Christian A; Klapschinski, Tim A; Janusko, Aron; Dickschat, Jeroen S

    2015-11-01

    An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product. PMID:26361082

  12. Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

    PubMed

    Rabe, Patrick; Barra, Lena; Rinkel, Jan; Riclea, Ramona; Citron, Christian A; Klapschinski, Tim A; Janusko, Aron; Dickschat, Jeroen S

    2015-11-01

    An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.

  13. 13C Tracking after 13CO2 Supply Revealed Diurnal Patterns of Wood Formation in Aspen1

    PubMed Central

    Mahboubi, Amir; Linden, Pernilla; Moritz, Thomas

    2015-01-01

    Wood of trees is formed from carbon assimilated in the photosynthetic tissues. Determining the temporal dynamics of carbon assimilation, subsequent transport into developing wood, and incorporation to cell walls would further our understanding of wood formation in particular and tree growth in general. To investigate these questions, we designed a 13CO2 labeling system to study carbon transport and incorporation to developing wood of hybrid aspen (Populus tremula × tremuloides). Tracking of 13C incorporation to wood over a time course using nuclear magnetic resonance spectroscopy revealed diurnal patterns in wood cell wall biosynthesis. The dark period had a differential effect on 13C incorporation to lignin and cell wall carbohydrates. No 13C was incorporated into aromatic amino acids of cell wall proteins in the dark, suggesting that cell wall protein biosynthesis ceased during the night. The results show previously unrecognized temporal patterns in wood cell wall biosynthesis, suggest diurnal cycle as a possible cue in the regulation of carbon incorporation to wood, and establish a unique 13C labeling method for the analysis of wood formation and secondary growth in trees. PMID:25931520

  14. Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study

    PubMed Central

    1989-01-01

    We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers

  15. In vivo dynamic turnover of cerebral 13C isotopomers from [U- 13C]glucose

    NASA Astrophysics Data System (ADS)

    Xu, Su; Shen, Jun

    2006-10-01

    An INEPT-based 13C MRS method and a cost-effective and widely available 11.7 Tesla 89-mm bore vertical magnet were used to detect dynamic 13C isotopomer turnover from intravenously infused [U- 13C]glucose in a 211 μL voxel located in the adult rat brain. The INEPT-based 1H → 13C polarization transfer method is mostly adiabatic and therefore minimizes signal loss due to B 1 inhomogeneity of the surface coils used. High quality and reproducible data were acquired as a result of combined use of outer volume suppression, ISIS, and the single-shot three-dimensional localization scheme built in the INEPT pulse sequence. Isotopomer patterns of both glutamate C4 at 34.00 ppm and glutamine C4 at 31.38 ppm are dominated first by a doublet originated from labeling at C4 and C5 but not at C3 (with 1JC4C5 = 51 Hz) and then by a quartet originated from labeling at C3, C4, and C5 (with 1JC3C4 = 35 Hz). A lag in the transition of glutamine C4 pattern from doublet-dominance to quartet dominance as compared to glutamate C4 was observed, which provides an independent verification of the precursor-product relationship between neuronal glutamate and glial glutamine and a significant intercompartmental cerebral glutamate-glutamine cycle between neurons and glial cells.

  16. Synthesis and applications of {sup 13}C glycerol

    SciTech Connect

    Stocking, E.; Khalsa, O.; Martinez, R.A.; Silks, L.A. III

    1994-12-01

    Due in part to the use of labeled glycerol for the {sup 13}C enrichment of biomolecules, we are currently developing new synthetic routes to various isotopomers of glycerol. Judging from our experience, traditional methods of glycerol synthesis are not easily adapted for isotopic enrichment and/or have poor overall yields (12 to 15%). Furthermore, the use of glycerol for enrichment can be prohibitively expensive and its availability is limited by the level of demand. We are presently developing a short de novo synthesis of glycerol from carbon dioxide ({approximately}53% overall yield for four steps) and are examining the feasibility of synthesizing site-specific {sup 13}C-labeled glycerol and dihydroxyacetone (DHA) from labeled methanol and carbon dioxide. One application of {sup 13}C glycerol we have examined is enzymatic conversion of glycerol to glyceraldehyde-3-monophosphate or dihydroxyacetone monophosphate (DHAP) with yields ranging from 25 to 50% (as determined by NMR spectroscopy). We are also pursuing the chemical conversion of {sup 13}C-labeled DHA to DHAP. We are especially interested in {sup 13}C-labeled DHAP because we are investigating its use as a chemo-enzymatic precursor for both labeled 2-deoxyribose and 2-deoxyribonucleic acids.

  17. INCORPORATION OF LABELED NITRIC OXIDE INTO RESPIRATORY TRACT LINING FLUIDS AND BLOOD PLASMA DURING LUNG INFLAMMATION

    EPA Science Inventory

    Incorporation of labeled nitric oxide (N18O) into respiratory tract lining fluids and blood plasma during lung inflammation. Slade, R., Norwood, J., Crissman, K., McKee, J., Hatch, G. PTB, ETD, NHEERL, ORD, USEPA, Res. Tri. Pk., NC

    Our earlier studies have demonstrated t...

  18. SIMS measurements of intrashell δ13C in the cultured planktic foraminifer Orbulina universa

    NASA Astrophysics Data System (ADS)

    Vetter, Lael; Kozdon, Reinhard; Valley, John W.; Mora, Claudia I.; Spero, Howard J.

    2014-08-01

    In this study, we present experimental results from the planktic foraminifer Orbulina universa, cultured in the laboratory. We demonstrate that the δ13C of shell calcite precipitated in 13C-labeled seawater for 24 h can be resolved and accurately measured using Secondary Ion Mass Spectrometry (SIMS). Specimens maintained at 20 °C were transferred from ambient seawater (δ13CDIC = +1.3‰) into seawater with δ13CDIC = +51.5‰ and enriched [Ba2+] for 24 h. Specimens were then transferred into ambient seawater with elevated [87Sr] for 6-9 h of calcification, followed by a transfer back into unlabeled ambient seawater until gametogenesis. This technique produced O. universa shells with calcite layers of distinct geochemical signatures. We quantify the spatial positions of trace element labels in the shells using laser ablation ICP-MS depth profiling. Using fragments from the same shells, we quantify intrashell δ13Ccalcite using SIMS with a 6 or 8 μm spot (2 SD range ±0.5‰ to 1.7‰). Measured δ13Ccalcite values in O. universa shell layers precipitated in ambient seawater are within 2‰ of predicted δ13Ccalcite values. In 13C-labeled bands of calcite, 6 μm SIMS spot measurements are within 2‰ of predicted δ13Ccalcite values, whereas 8 μm SIMS spots yield intermediate, mixed values. The spatial agreement between trace element and carbon isotope data suggests that 13C and cation tracers are synchronously incorporated into shell calcite. These results demonstrate the ability of SIMS δ13C measurements to resolve ∼10 μm features in foraminifer shell calcite using a 6 μm spot, and highlight the potential of this technique for addressing questions about ecology, biomineralization, and paleoceanography.

  19. A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme

    NASA Astrophysics Data System (ADS)

    Lv, Guohua; Faßhuber, Hannes Klaus; Loquet, Antoine; Demers, Jean-Philippe; Vijayan, Vinesh; Giller, Karin; Becker, Stefan; Lange, Adam

    2013-03-01

    The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-13C]Glucose ([2-13C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-13C]pyruvate produced mainly by glycolysis and [3-13C]/[1,3-13C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs 13Cβ-13Cγ2 and 13Cα-13Cγ1 in Val, and 13Cγ-13Cδ2 and 13Cβ-13Cδ1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin.

  20. Synthesis Of [2h, 13c]M [2h2m 13c], And [2h3,, 13c] Methyl Aryl Sulfones And Sulfoxides

    DOEpatents

    Martinez, Rodolfo A.; Alvarez, Marc A.; Silks, III, Louis A.; Unkefer, Clifford J.; Schmidt, Jurgen G.

    2004-07-20

    The present invention is directed to labeled compounds, [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2, .sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfones and [.sup.2 H.sub.1, .sup.13 C], [.sup.2 H.sub.2, .sup.13 C] and [.sup.2 H.sub.3, .sup.13 C]methyl aryl sulfoxides, wherein the .sup.13 C methyl group attached to the sulfur of the sulfone or sulfoxide includes exactly one, two or three deuterium atoms and the aryl group is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure: ##STR1## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently, hydrogen, a C.sub.1 -C.sub.4 lower alkyl, a halogen, an amino group from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each a C.sub.1 -C.sub.4 lower alkyl, a phenyl, or an alkoxy group. The present invention is also directed to processes of preparing methyl aryl sulfones and methyl aryl sulfoxides.

  1. Approaches to studies on neuronal/glial relationships by 13C-MRS analysis.

    PubMed

    Taylor, A; McLean, M; Morris, P; Bachelard, H

    1996-01-01

    The use of different 13C-labelled precursors alone or in combination ([1-13C]glucose, [2-13C]glucose, [1-13C]acetate, [2-13C]acetate and [1,2-13C2]acetate) to study neuronal/glial metabolic relationships by MRS is discussed. Glutamine and citrate resonances represent glial metabolism if a combination of [1-13C]glucose + [2-13C]acetate is used, but only for short time periods. A combination of [2-13C]glucose + [2-13C]acetate will label -COO- groups from glucose and -CH2 groups from acetate, respectively, which distinguish well in theory. However, this approach is severely limited by the long T1S of -COO- groups and low S/N. Contributions of the anaplerotic pathway can be assessed using [2-13C]glucose, but again can be limited by the long T1S of -COO- groups. Labelling of glycerol-3-phosphate (believed to be produced in glia) from [1-13C]glucose is difficult to see under normal conditions but has proved useful in, e.g., hypoxia. We believe the most promising approach is the use of [1-13C] glucose with [1,2-13C2]acetate, by analysis of the multiplets ('isotopomers') of the amino acid resonances.

  2. Measuring DNA synthesis rates with [1-13C]glycine.

    PubMed

    Chen, P; Abramson, F P

    1998-05-01

    We have devised and evaluated a stable-isotopic method for measuring DNA synthesis rates. The probe is [1-13C]-glycine that is incorporated into purines via de novo biosynthesis. The human hepatoma cell line HEP G2 was grown in medium containing [1-13C]glycine, the cells were harvested at various times, and the DNA was extracted. Following hydrolysis to the nucleosides, a reversed-phase HPLC separation was used to provide separate peaks for deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The HPLC effluent was continuously fed into a chemical reaction interface and an isotope ratio mass spectrometer (HPLC/CRI/IRMS). The isotope ratio of the CO2 produced in the CRI was used to monitor for enrichment. The cells were grown continuously for 5 days in labeled medium and also in a 1-day pulse labeling experiment where the washout of label was observed for the subsequent 9 days. As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13C/12C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1-13C]-glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer. PMID:9599574

  3. Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation

    SciTech Connect

    Packham, M.A.; Guccione, M.A.; Bryant, N.L.; Livne, A. )

    1990-07-01

    Previous experiments showed that during the early stages (20-30 seconds) of aggregation induced by adenosine diphosphate (ADP, 2 microM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with (3H)palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA (ethylene glycol-bis-(beta-aminoethyl ether))-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated cytoskeletons. In both species, and with either ADP or thrombin as aggregating agent, 70-85% of the label in both intact platelets and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets. The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and palmitic acid into the cytoskeleton.

  4. A simple method for the measurement of labelled compound incorporation into cells in culture.

    PubMed

    Suplisson, A; Boissel, J P

    1976-02-10

    A simple method for the measurement of labelled compound incorporation into cells in layer culture was developed. Compared to other methods it proves to spare time and to be more sensitive owing to the fact that cells are not detached from the culture vials until the end of the manipulation as these are dissolved in the scintillation medium together with the cells just before counting.

  5. /sup 13/C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and of the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

    SciTech Connect

    Cohen, S.M.

    1987-01-27

    /sup 13/C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the /sup 13/C enrichments at the individual carbons of glutamate when (3-/sup 13/C)alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of /sup 13/C label into fatty acids was monitored in this liver preparation. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by (1-/sup 13/C)acetyl-CoA (from (2-/sup 13/C)pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by (3-/sup 13/C)alanine plus (2-/sup 13/C)ethanol, which are converted to (2-/sup 13/C)acetyl-CoA. Thus, measurement of /sup 13/C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids. In addition to obtaining the rate of lipogenesis, it was possible to distinguish the contributions of chain elongation from those of the de novo synthesis pathway and to estimate the average chain length of the /sup 13/C-labeled fatty acids produced.

  6. 13C-based metabolic flux analysis: fundamentals and practice.

    PubMed

    Yang, Tae Hoon

    2013-01-01

    Isotope-based metabolic flux analysis is one of the emerging technologies applied to system level metabolic phenotype characterization in metabolic engineering. Among the developed approaches, (13)C-based metabolic flux analysis has been established as a standard tool and has been widely applied to quantitative pathway characterization of diverse biological systems. To implement (13)C-based metabolic flux analysis in practice, comprehending the underlying mathematical and computational modeling fundamentals is of importance along with carefully conducted experiments and analytical measurements. Such knowledge is also crucial when designing (13)C-labeling experiments and properly acquiring key data sets essential for in vivo flux analysis implementation. In this regard, the modeling fundamentals of (13)C-labeling systems and analytical data processing are the main topics we will deal with in this chapter. Along with this, the relevant numerical optimization techniques are addressed to help implementation of the entire computational procedures aiming at (13)C-based metabolic flux analysis in vivo.

  7. A scientific workflow framework for (13)C metabolic flux analysis.

    PubMed

    Dalman, Tolga; Wiechert, Wolfgang; Nöh, Katharina

    2016-08-20

    Metabolic flux analysis (MFA) with (13)C labeling data is a high-precision technique to quantify intracellular reaction rates (fluxes). One of the major challenges of (13)C MFA is the interactivity of the computational workflow according to which the fluxes are determined from the input data (metabolic network model, labeling data, and physiological rates). Here, the workflow assembly is inevitably determined by the scientist who has to consider interacting biological, experimental, and computational aspects. Decision-making is context dependent and requires expertise, rendering an automated evaluation process hardly possible. Here, we present a scientific workflow framework (SWF) for creating, executing, and controlling on demand (13)C MFA workflows. (13)C MFA-specific tools and libraries, such as the high-performance simulation toolbox 13CFLUX2, are wrapped as web services and thereby integrated into a service-oriented architecture. Besides workflow steering, the SWF features transparent provenance collection and enables full flexibility for ad hoc scripting solutions. To handle compute-intensive tasks, cloud computing is supported. We demonstrate how the challenges posed by (13)C MFA workflows can be solved with our approach on the basis of two proof-of-concept use cases.

  8. A scientific workflow framework for (13)C metabolic flux analysis.

    PubMed

    Dalman, Tolga; Wiechert, Wolfgang; Nöh, Katharina

    2016-08-20

    Metabolic flux analysis (MFA) with (13)C labeling data is a high-precision technique to quantify intracellular reaction rates (fluxes). One of the major challenges of (13)C MFA is the interactivity of the computational workflow according to which the fluxes are determined from the input data (metabolic network model, labeling data, and physiological rates). Here, the workflow assembly is inevitably determined by the scientist who has to consider interacting biological, experimental, and computational aspects. Decision-making is context dependent and requires expertise, rendering an automated evaluation process hardly possible. Here, we present a scientific workflow framework (SWF) for creating, executing, and controlling on demand (13)C MFA workflows. (13)C MFA-specific tools and libraries, such as the high-performance simulation toolbox 13CFLUX2, are wrapped as web services and thereby integrated into a service-oriented architecture. Besides workflow steering, the SWF features transparent provenance collection and enables full flexibility for ad hoc scripting solutions. To handle compute-intensive tasks, cloud computing is supported. We demonstrate how the challenges posed by (13)C MFA workflows can be solved with our approach on the basis of two proof-of-concept use cases. PMID:26721184

  9. FITC-labeled lipopolysaccharide: use as a probe for liposomal membrane incorporation studies.

    PubMed

    Trubetskoy, V S; Koshkina, N V; Omel'yanenko, V G; L'vov, V L; Dmitriev, B A; Petrov, A B; Torchilin, V P

    1990-08-20

    FITC-labeled LPS from Neisseria meningitidis can be used as a probe to follow the process of LPS incorporation into liposomal membrane and to study its interaction with a bilayer. The incorporation of FITC-LPS into the bilayer was proved by physicochemical methods as well as by liposomal LPS toxicity decrease in actinomycin D-sensitized mice. Fluorescence intensity increase was observed upon the insertion of FITC-LPS into the membrane of dehydration/rehydration vesicles and vesicles obtained by co-sonication of lipid suspension and FITC-LPS. Following FITC-LPS fluorescence polarization it was shown that the substance seems to be clusterized in the liposomal membrane starting from FITC-LPS/lipid molar ratio 1:800.

  10. Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid delta13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry.

    PubMed

    Dungait, Jennifer A J; Briones, Maria J I; Bol, Roland; Evershed, Richard P

    2008-06-01

    Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.

  11. A study on the biosynthesis of hygrophorone B(12) in the mushroom Hygrophorus abieticola reveals an unexpected labelling pattern in the cyclopentenone moiety.

    PubMed

    Otto, Alexander; Porzel, Andrea; Schmidt, Jürgen; Wessjohann, Ludger; Arnold, Norbert

    2015-10-01

    The hitherto unknown natural formation of hygrophorones, antibacterial and antifungal cyclopentenone derivatives from mushrooms, was investigated for hygrophorone B(12) in Hygrophorus abieticola Krieglst. ex Gröger & Bresinsky by feeding experiments in the field using (13)C labelled samples of D-glucose and sodium acetate. The incorporation of (13)C isotopes was extensively studied using 1D and 2D NMR spectroscopy as well as ESI-HRMS analyses. In the experiment with [U-(13)C6]-glucose, six different (13)C2 labelled isotopomers were observed in the 2D INADEQUATE spectrum due to incorporation of [1,2-(13)C2]-acetyl-CoA. This labelling pattern demonstrated that hygrophorone B(12) is derived from a fatty acid-polyketide route instead of a 1,4-α-D-glucan derived anhydrofructose pathway. The experiment with [2-(13)C]-acetate revealed an unexpected incorporation pattern in the cyclopentenone functionality of hygrophorone B(12). Four single-labelled isotopomers, in particular [1-(13)C]-, [2-(13)C]-, [3-(13)C]-, and [4-(13)C]-hygrophorone B(12), were detected that showed only half enrichment in comparison to the respective labelled alkyl side chain carbons. This labelling pattern indicates the formation of a symmetrical intermediate during hygrophorone B(12) biosynthesis. Based on these observations, a biogenetic route via a 4-oxo fatty acid and a chrysotrione B homologue is discussed.

  12. 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.

  13. Tracing carbon assimilation in endosymbiotic deep-sea hydrothermal vent Mytilid fatty acids by 13C-fingerprinting

    NASA Astrophysics Data System (ADS)

    Riou, V.; Bouillon, S.; Serrão Santos, R.; Dehairs, F.; Colaço, A.

    2010-09-01

    Bathymodiolus azoricus mussels thrive at Mid-Atlantic Ridge hydrothermal vents, where part of their energy requirements are met via an endosymbiotic association with chemolithotrophic and methanotrophic bacteria. In an effort to describe phenotypic characteristics of the two bacterial endosymbionts and to assess their ability to assimilate CO2, CH4 and multi-carbon compounds, we performed experiments in aquaria using 13C-labeled NaHCO3 (in the presence of H2S), CH4 or amino-acids and traced the incorporation of 13C into total and phospholipid fatty acids (tFA and PLFA, respectively). 14:0; 15:0; 16:0; 16:1(n - 7)c+t; 18:1(n - 13)c+t and (n - 7)c+t; 20:1(n - 7); 20:2(n - 9,15); 18:3(n - 7) and (n - 5,10,13) PLFA were labeled in the presence of H13CO3- (+H2S) and 13CH4, while the 12:0 compound became labeled only in the presence of H13CO3- (+H2S). In contrast, the 17:0; 18:0; 16:1(n - 9); 16:1(n - 8) and (n - 6); 18:1(n - 8); and 18:2(n - 7) PLFA were only labeled in the presence of 13CH4. Some of these symbiont-specific fatty acids also appeared to be labeled in mussel gill tFA when incubated with 13C-enriched amino acids, and so were mussel-specific fatty acids such as 22:2(n - 7,15). Our results provide experimental evidence for the potential of specific fatty acid markers to distinguish between the two endosymbiotic bacteria, shedding new light on C1 and multi-carbon compound metabolic pathways in B. azoricus and its symbionts.

  14. Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1-13C]butyrate and [1-13C]pyruvate

    PubMed Central

    Bastiaansen, Jessica A. M.; Merritt, Matthew E.; Comment, Arnaud

    2016-01-01

    Cardiac dysfunction is often associated with a shift in substrate preference for ATP production. Hyperpolarized (HP) 13C magnetic resonance spectroscopy (MRS) has the unique ability to detect real-time metabolic changes in vivo due to its high sensitivity and specificity. Here a protocol using HP [1-13C]pyruvate and [1-13C]butyrate is used to measure carbohydrate versus fatty acid metabolism in vivo. Metabolic changes in fed and fasted Sprague Dawley rats (n = 36) were studied at 9.4 T after tail vein injections. Pyruvate and butyrate competed for acetyl-CoA production, as evidenced by significant changes in [13C]bicarbonate (−48%), [1-13C]acetylcarnitine (+113%), and [5-13C]glutamate (−63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-13C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-13C]acetoacetate and [1-13C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-13C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (−82%). Combining HP 13C technology and co-administration of separate imaging agents enables noninvasive and simultaneous monitoring of both fatty acid and carbohydrate oxidation. This protocol illustrates a novel method for assessing metabolic flux through different enzymatic pathways simultaneously and enables mechanistic studies of the changing myocardial energetics often associated with disease. PMID:27150735

  15. Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1-(13)C]butyrate and [1-(13)C]pyruvate.

    PubMed

    Bastiaansen, Jessica A M; Merritt, Matthew E; Comment, Arnaud

    2016-01-01

    Cardiac dysfunction is often associated with a shift in substrate preference for ATP production. Hyperpolarized (HP) (13)C magnetic resonance spectroscopy (MRS) has the unique ability to detect real-time metabolic changes in vivo due to its high sensitivity and specificity. Here a protocol using HP [1-(13)C]pyruvate and [1-(13)C]butyrate is used to measure carbohydrate versus fatty acid metabolism in vivo. Metabolic changes in fed and fasted Sprague Dawley rats (n = 36) were studied at 9.4 T after tail vein injections. Pyruvate and butyrate competed for acetyl-CoA production, as evidenced by significant changes in [(13)C]bicarbonate (-48%), [1-(13)C]acetylcarnitine (+113%), and [5-(13)C]glutamate (-63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-(13)C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-(13)C]acetoacetate and [1-(13)C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-(13)C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (-82%). Combining HP (13)C technology and co-administration of separate imaging agents enables noninvasive and simultaneous monitoring of both fatty acid and carbohydrate oxidation. This protocol illustrates a novel method for assessing metabolic flux through different enzymatic pathways simultaneously and enables mechanistic studies of the changing myocardial energetics often associated with disease. PMID:27150735

  16. Comparison of labeled acetate and glucose incorporations into lipids in the liver and adipose tissue after intravenous injection in rats.

    PubMed

    Iritani, Nobuko; Hirakawa, Tomoe; Fukuda, Hitomi; Katsukawa, Michiko; Kouno, Mika

    2014-01-01

    To compare incorporations of acetate and glucose in tissue total lipids and triacylglycerols (TAG), incorporations of labeled acetate and glucose in livers and epididymal adipose tissues (adipose tissue) were followed after their intravenous injection in the tail vein of individual rat fed a fat-free or 10% corn oil diet. The incorporation of acetate into total lipids (mostly TAG) in the liver reached maximum 2 h after the injection, while the incorporation of glucose decreased more quickly. Incorporation of glucose into total lipids and TAG was more greatly suppressed by dietary corn oil than that of acetate in the liver. In the adipose tissues, the incorporation of labeled acetate or glucose into total lipids was maximum 2-8 h after the injection, while the incorporation of glucose was very low, especially in rats fed the corn oil diet. Moreover, the time courses for labeled acetate and glucose incorporations into total lipids in the liver were parallel to those in plasma, but opposite to those in adipose tissue. TAG synthesized from acetate and glucose in the liver appeared to be mostly transported to adipose tissue. Thus, it is suggested that as the labeled glucose rapidly decreased in the liver, plasma and adipose tissue, TAG should be less derived from dietary carbohydrate than from dietary fat.

  17. Stable isotope labeling, in vivo, of D- and L-tryptophan pools in lemna gibba and the low incorporation of label into indole-3-acetic acid

    SciTech Connect

    Baldi, B.G. ); Maher, B.R. ); Slovin, J.P.; Cohen, J.D. Univ. of Maryland, College Park )

    1991-04-01

    The authors present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of ({sup 15}N-indole)-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of L-({sup 15}N)tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled L-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into D-tryptophan. D-({sup 15}N)trytophan supplied to Lemna at rates of approximately 400 times excess of endogenous D-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of L-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that L-tryptophan is a more direct precursor to IAA than the D isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that L-tryptophan also may not be a primary precursor to IAA in plants.

  18. Sucrose octabenzoate: assignment of 13C and 1H resonances of the sucrose moiety and the 13C resonances of the carbonyl carbons. Use of 13C-n.m.r. spectroscopy for the study of selective deacylation.

    PubMed

    Colquhoun, I J; Haines, A H; Konowicz, P A; Jones, H F

    1990-09-19

    Assignment of the 1H and 13C signals arising from the carbohydrate portion of sucrose octabenzoate has been achieved using homonuclear shift correlation experiments (COSY) and one-bond 1H-13C heteronuclear shift correlation measurements, respectively. The 13C resonances of the carbonyl carbon atoms of the eight benzoyl groups are readily distinguished for solutions in benzene-d6-pyridine-d5 (1:1), and have been assigned by means of three-bond 1H-13C shift correlation studies coupled with measurement of the 13C-n.m.r. spectrum of a sucrose octabenzoate specifically labelled with 13C in some of the carbonyl groups. With this assignment, products of partial deacylation of the octabenzoate may readily be identified by treatment with excess of benzoyl-carbonyl-13C chloride followed by measurement of the 13C-n.m.r. spectrum of the labelled sucrose octabenzoate, so prepared, in the carbonyl region. PMID:2276151

  19. Imaging the time-integrated cerebral metabolic activity with subcellular resolution through nanometer-scale detection of biosynthetic products deriving from (13)C-glucose.

    PubMed

    Takado, Yuhei; Knott, Graham; Humbel, Bruno M; Masoodi, Mojgan; Escrig, Stéphane; Meibom, Anders; Comment, Arnaud

    2015-11-01

    Glucose is the primary source of energy for the brain but also an important source of building blocks for proteins, lipids, and nucleic acids. Little is known about the use of glucose for biosynthesis in tissues at the cellular level. We demonstrate that local cerebral metabolic activity can be mapped in mouse brain tissue by quantitatively imaging the biosynthetic products deriving from [U-(13)C]glucose metabolism using a combination of in situ electron microscopy and secondary ion mass-spectroscopy (NanoSIMS). Images of the (13)C-label incorporated into cerebral ultrastructure with ca. 100 nm resolution allowed us to determine the timescale on which the metabolic products of glucose are incorporated into different cells, their sub-compartments and organelles. These were mapped in astrocytes and neurons in the different layers of the motor cortex. We see evidence for high metabolic activity in neurons via the nucleus (13)C enrichment. We observe that in all the major cell compartments, such as e.g. nucleus and Golgi apparatus, neurons incorporate substantially higher concentrations of (13)C-label than astrocytes. PMID:26409162

  20. Application of (13)C-stable isotope probing to identify RDX-degrading microorganisms in groundwater.

    PubMed

    Cho, Kun-Ching; Lee, Do Gyun; Roh, Hyungkeun; Fuller, Mark E; Hatzinger, Paul B; Chu, Kung-Hui

    2013-07-01

    We employed stable isotope probing (SIP) with (13)C-labeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to identify active microorganisms responsible for RDX biodegradation in groundwater microcosms. Sixteen different 16S rRNA gene sequences were derived from microcosms receiving (13)C-labeled RDX, suggesting the presence of microorganisms able to incorporate carbon from RDX or its breakdown products. The clones, residing in Bacteroidia, Clostridia, α-, β- and δ-Proteobacteria, and Spirochaetes, were different from previously described RDX degraders. A parallel set of microcosms was amended with cheese whey and RDX to evaluate the influence of this co-substrate on the RDX-degrading microbial community. Cheese whey stimulated RDX biotransformation, altered the types of RDX-degrading bacteria, and decreased microbial community diversity. Results of this study suggest that RDX-degrading microorganisms in groundwater are more phylogenetically diverse than what has been inferred from studies with RDX-degrading isolates. PMID:23603473

  1. New guidelines for δ13C measurements

    USGS Publications Warehouse

    Coplen, Tyler B.; Brand, Willi A.; Gehre, Matthias; Groning, Manfred; Meijer, Harro A. J.; Toman, Blaza; Verkouteren, R. Michael

    2006-01-01

    Consistency of δ13C measurements can be improved 39−47% by anchoring the δ13C scale with two isotopic reference materials differing substantially in 13C/12C. It is recommended thatδ13C values of both organic and inorganic materials be measured and expressed relative to VPDB (Vienna Peedee belemnite) on a scale normalized by assigning consensus values of −46.6‰ to L-SVEC lithium carbonate and +1.95‰ to NBS 19 calcium carbonate. Uncertainties of other reference material values on this scale are improved by factors up to two or more, and the values of some have been notably shifted:  the δ13C of NBS 22 oil is −30.03%.

  2. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  3. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.

    PubMed

    Evans, Eric G B; Millhauser, Glenn L

    2015-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents

  4. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.

    PubMed

    Evans, Eric G B; Millhauser, Glenn L

    2015-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents

  5. A comparison of quantitative methods for clinical imaging with hyperpolarized (13)C-pyruvate.

    PubMed

    Daniels, Charlie J; McLean, Mary A; Schulte, Rolf F; Robb, Fraser J; Gill, Andrew B; McGlashan, Nicholas; Graves, Martin J; Schwaiger, Markus; Lomas, David J; Brindle, Kevin M; Gallagher, Ferdia A

    2016-04-01

    Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data.

  6. Synthesis of an Isotopically Labeled Naphthalene Derivative That Supports a Long-Lived Nuclear Singlet State

    PubMed Central

    2015-01-01

    The synthesis of an octa-alkoxy substituted isotopically labeled naphthalene derivative, shown to have excellent properties in singlet NMR experiments, is described. This highly substituted naphthalene system, which incorporates an adjacent 13C spin pair, is readily accessed from a commercially available 13C2-labeled building block via sequential thermal alkynyl- and arylcyclobutenone rearrangements. The synthetic route incorporates a simple desymmetrization approach leading to a small difference in the chemical shifts of the 13C spin pair, a design constraint crucial for accessing nuclear singlet order. PMID:25898076

  7. Tracing carbon assimilation in endosymbiotic deep-sea hydrothermal vent Mytilid fatty acids by 13C-fingerprinting

    NASA Astrophysics Data System (ADS)

    Riou, V.; Bouillon, S.; Serrão Santos, R.; Dehairs, F.; Colaço, A.

    2010-05-01

    Bathymodiolus azoricus mussels thrive at Mid-Atlantic Ridge hydrothermal vents, where part of their energy requirements are met via an endosymbiotic association with chemolithotrophic and methanotrophic bacteria. In an effort to describe phenotypic characteristics of the two bacterial endosymbionts and to assess their ability to assimilate CO2, CH4 and multi-carbon compounds, we performed experiments in aquaria using 13C-labeled NaHCO3 (in the presence of H2S), CH4 or amino-acids and traced the incorporation of 13C into total and phospholipid fatty acids (tFA and PLFA, respectively). 14:0, 15:0, 16:1(n-7)c+t and 18:1(n-7)c+t PLFA were labeled in the presence of H13CO3- (+H2S) and 13CH4, while the 12:0 compound became labeled only in the presence of H13CO3- (+H2S). In contrast, the 16:1(n-9), 16:1(n-8) and (n-6), 18:1(n-8)c and (n-7), 20:1(n-7) and 18:2(n-7) PLFA were only labeled in the presence of 13CH4. Some of these symbiont-specific fatty acids also appeared to be labeled in mussel gill tFA when incubated with 13C-enriched amino acids, and so were mussel-specific fatty acids such as 22:2(n-7,15). Our results provide experimental evidence for the potential of specific fatty acid markers to distinguish between the two endosymbiotic bacteria, shedding new light on C1 and multi-carbon compound metabolic pathways in B. azoricus and its symbionts.

  8. OpenMebius: An Open Source Software for Isotopically Nonstationary 13C-Based Metabolic Flux Analysis

    PubMed Central

    Furusawa, Chikara

    2014-01-01

    The in vivo measurement of metabolic flux by 13C-based metabolic flux analysis (13C-MFA) provides valuable information regarding cell physiology. Bioinformatics tools have been developed to estimate metabolic flux distributions from the results of tracer isotopic labeling experiments using a 13C-labeled carbon source. Metabolic flux is determined by nonlinear fitting of a metabolic model to the isotopic labeling enrichment of intracellular metabolites measured by mass spectrometry. Whereas 13C-MFA is conventionally performed under isotopically constant conditions, isotopically nonstationary 13C metabolic flux analysis (INST-13C-MFA) has recently been developed for flux analysis of cells with photosynthetic activity and cells at a quasi-steady metabolic state (e.g., primary cells or microorganisms under stationary phase). Here, the development of a novel open source software for INST-13C-MFA on the Windows platform is reported. OpenMebius (Open source software for Metabolic flux analysis) provides the function of autogenerating metabolic models for simulating isotopic labeling enrichment from a user-defined configuration worksheet. Analysis using simulated data demonstrated the applicability of OpenMebius for INST-13C-MFA. Confidence intervals determined by INST-13C-MFA were less than those determined by conventional methods, indicating the potential of INST-13C-MFA for precise metabolic flux analysis. OpenMebius is the open source software for the general application of INST-13C-MFA. PMID:25006579

  9. OpenMebius: an open source software for isotopically nonstationary 13C-based metabolic flux analysis.

    PubMed

    Kajihata, Shuichi; Furusawa, Chikara; Matsuda, Fumio; Shimizu, Hiroshi

    2014-01-01

    The in vivo measurement of metabolic flux by (13)C-based metabolic flux analysis ((13)C-MFA) provides valuable information regarding cell physiology. Bioinformatics tools have been developed to estimate metabolic flux distributions from the results of tracer isotopic labeling experiments using a (13)C-labeled carbon source. Metabolic flux is determined by nonlinear fitting of a metabolic model to the isotopic labeling enrichment of intracellular metabolites measured by mass spectrometry. Whereas (13)C-MFA is conventionally performed under isotopically constant conditions, isotopically nonstationary (13)C metabolic flux analysis (INST-(13)C-MFA) has recently been developed for flux analysis of cells with photosynthetic activity and cells at a quasi-steady metabolic state (e.g., primary cells or microorganisms under stationary phase). Here, the development of a novel open source software for INST-(13)C-MFA on the Windows platform is reported. OpenMebius (Open source software for Metabolic flux analysis) provides the function of autogenerating metabolic models for simulating isotopic labeling enrichment from a user-defined configuration worksheet. Analysis using simulated data demonstrated the applicability of OpenMebius for INST-(13)C-MFA. Confidence intervals determined by INST-(13)C-MFA were less than those determined by conventional methods, indicating the potential of INST-(13)C-MFA for precise metabolic flux analysis. OpenMebius is the open source software for the general application of INST-(13)C-MFA.

  10. Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells

    PubMed Central

    Metallo, Christian M.; Walther, Jason L.; Stephanopoulos, Gregory

    2009-01-01

    13C metabolic flux analysis (MFA) is the most comprehensive means of characterizing cellular metabolic states. Uniquely labeled isotopic tracers enable more focused analyses to probe specific reactions within the network. As a result, the choice of tracer largely determines the precision with which one can estimate metabolic fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled 13C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2-13C2]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2-13C]glucose and [3-13C]glucose also outperformed the more commonly used [1-13C]glucose. [U-13C5]glutamine emerged as the preferred isotopic tracer for analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on the performance of 13C-labeled substrates and can aid in the design of more informative MFA experiments in mammalian cell culture. PMID:19622376

  11. Metallic nanocrystallites-incorporated ordered mesoporous carbon as labels for a sensitive simultaneous multianalyte electrochemical immunoassay.

    PubMed

    Fang, Yishan; Huang, Xinjian; Zeng, Qiang; Wang, Lishi

    2015-11-15

    This work reports on a facile, novel multianalyte electrochemical immunoassay for simultaneous detection of a-fetoprotein (AFP) and human epidermal growth factor receptor type-2 (HER-2) using metal-containing nanomaterials confined in the ordered mesoporous carbon matrix (OMC-M) as labels. Well-dispersed uniform metallic nanocrystallites incorporated OMC materials were fabricated through a simple, economical, and green preparative strategy toward phenolic resol as a carbon source and metal nitrate as metal sources. The large amount of metallic nanocrystallites loading on the OMC nanomaterials, greatly amplified the detection signals, and the good biocompatibility of carbon nanotubes-chitosan retained excellent stability for the sandwich-type immunoassay. Under optimal experimental conditions, the proposed immunoassay exhibited high sensitivity and selectivity for the detection of analytes, providing a better linear response range from 0.001 to 150 ng/mL for AFP and for HER-2, with a lower limit of detectionof 0.6p g/mL and 0.35 pg/mL (S/N=3), respectively. The immunosensor exhibited convenience, low cost, rapidity, good specificity, acceptable stability and reproducibility. Moreover, satisfactory results were obtained for the determination of AFP and HER-2 in real human serum samples, indicating that the developed immunoassay has the potential to find application in clinical detection of AFP and HER-2 and other tumor markers as an alternative approach.

  12. An alternative and robust synthesis of [(13) C4 ]Baraclude® (entecavir).

    PubMed

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

    2013-10-01

    Stable isotope-labeled [(13) C4 ]entecavir (1) was prepared in 11 steps. Commercially available [(13) C]guanidine hydrochloride and diethyl[1,2,3-(13) C3 ]malonate were condensed to yield 2-amino[2,4,5,6-(13) C4 ]pyrimidine-4,6-diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [(13) C4 ]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be >99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0], <0.1%.

  13. In vivo13C spectroscopy in the rat brain using hyperpolarized [1- 13C]pyruvate and [2- 13C]pyruvate

    NASA Astrophysics Data System (ADS)

    Marjańska, Małgorzata; Iltis, Isabelle; Shestov, Alexander A.; Deelchand, Dinesh K.; Nelson, Christopher; Uğurbil, Kâmil; Henry, Pierre-Gilles

    2010-10-01

    The low sensitivity of 13C spectroscopy can be enhanced using dynamic nuclear polarization. Detection of hyperpolarized [1- 13C]pyruvate and its metabolic products has been reported in kidney, liver, and muscle. In this work, the feasibility of measuring 13C signals of hyperpolarized 13C metabolic products in the rat brain in vivo following the injection of hyperpolarized [1- 13C]pyruvate and [2- 13C]pyruvate is investigated. Injection of [2- 13C]pyruvate led to the detection of [2- 13C]lactate, but no other downstream metabolites such as TCA cycle intermediates were detected. Injection of [1- 13C]pyruvate enabled the detection of both [1- 13C]lactate and [ 13C]bicarbonate. A metabolic model was used to fit the hyperpolarized 13C time courses obtained during infusion of [1- 13C]pyruvate and to determine the values of VPDH and VLDH.

  14. Synthesis of [1-.sup.13C]pyruvic acid], [2-.sup.13C]pyruvic acid], [3-.sup.13C]pyruvic acid] and combinations thereof

    DOEpatents

    Martinez, Rodolfo A. , Unkefer; Clifford J. , Alvarez; Marc A.

    2012-06-12

    The present invention is directed to the labeled compounds, ##STR00001## wherein C* is each either .sup.13C and .sup.12C where at least one C* is .sup.13C, each hydrogen of the methylene group is hydrogen or deuterium, the methyl group includes either zero or three deuterium atoms, Q is sulfide, sulfinyl, or sulfone, Z is an aryl group such as 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, or a phenyl group ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently either hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group such as NH.sub.2, NHR and NRR' where R and R' are each independently either a C.sub.1-C.sub.4 lower alkyl, a phenyl, and an alkoxy group, and the methyl group can include either zero or three deuterium atoms. The present invention is also directed to the labeled compounds ##STR00003##

  15. Effects of trichloroacetic acid on the nitrogen metabolism of Pinus sylvestris--a 13C/15N tracer study.

    PubMed

    Hafner, Christoph; Jung, Klaus; Schüürmann, Gerrit

    2002-01-01

    Trichloroacetic acid (TCA) can be found in various environmental compartments like air, rain and plants all over the world. It is assumed that TCA is an atmospheric degradation product of volatile chloroorganic hydrocarbons. The herbicide effect of TCA in higher concentrations is well known, but not much is known about the phytotoxic effects in environmentally relevant concentrations. It can be shown in this study by using the 13C/15N stable isotope tracer technique that [13C]TCA is taken up by roots of two-year-old seedlings of Pinus sylvestris L. and transported into the needles. At the same time the effect of the substance on nitrogen metabolism can be analyzed by measuring the incorporation of 15NO3- into different nitrogen fractions of the plant. The more [13C]TCA incorporation, the higher the synthesis of 15N labelled amino acids and proteins is. These effects on the nitrogen metabolism are probably based on the activation of stress- and detoxification metabolism. It has to be assumed that there is an influence on N metabolism of Pinus sylvestris caused by the deposition of environmentally relevant TCA concentrations.

  16. A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

    PubMed Central

    Ghosh, Amit; Nilmeier, Jerome; Weaver, Daniel; Adams, Paul D.; Keasling, Jay D.; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Martín, Héctor García

    2014-01-01

    The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, 13C Metabolic Flux Analysis (13C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during 13C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of 13C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based 13C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species. PMID:25188426

  17. 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

  18. Compartmentation of glycogen metabolism revealed from 13C isotopologue distributions

    PubMed Central

    2011-01-01

    Background Stable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution. Results The software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells. Conclusions The analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast

  19. Incorporation of probabilistic seismic phase labels into a Bayesian multiple-event seismic locator

    SciTech Connect

    Myers, S; Johannesson, G; Hanley, W

    2008-01-17

    We add probabilistic phase labels to the multiple-event joint probability function of Myers et al., 2007 that formerly included event locations, travel-time corrections, and arrival-time measurement precision. Prior information on any of the multiple-event parameters may be used. The phase-label model includes a null label that captures phases not belonging to the collection of phases under consideration. Using the Markov-Chain Monte Carlo method, samples are drawn from the multiple-event joint probability function to infer the posteriori distribution that is consistent with priors and the arrival-time data set. Using this approach phase-label error can be accessed and phase-label error is propagated to all other multiple-event parameters. We test the method using a ground-truth data set of nuclear explosions at the Nevada Test Site. We find that posteriori phase labels agree with the meticulously analyzed data set in more than 97% of instances and the results are robust even when the input phase-label information is discarded. Only when a large percentage of the arrival-time data are corrupted does prior phase label information improve resolution of multiple-event parameters. Simultaneous modeling of the entire multiple-event system results in accurate posteriori probability regions for each multiple-event parameter.

  20. Bacteria and fungi respond differently to multifactorial climate change in a temperate heathland, traced with 13C-glycine and FACE CO2.

    PubMed

    Andresen, Louise C; Dungait, Jennifer A J; Bol, Roland; Selsted, Merete B; Ambus, Per; Michelsen, Anders

    2014-01-01

    It is vital to understand responses of soil microorganisms to predicted climate changes, as these directly control soil carbon (C) dynamics. The rate of turnover of soil organic carbon is mediated by soil microorganisms whose activity may be affected by climate change. After one year of multifactorial climate change treatments, at an undisturbed temperate heathland, soil microbial community dynamics were investigated by injection of a very small concentration (5.12 µg C g(-1) soil) of (13)C-labeled glycine ((13)C2, 99 atom %) to soils in situ. Plots were treated with elevated temperature (+1°C, T), summer drought (D) and elevated atmospheric carbon dioxide (510 ppm [CO2]), as well as combined treatments (TD, TCO2, DCO2 and TDCO2). The (13)C enrichment of respired CO2 and of phospholipid fatty acids (PLFAs) was determined after 24 h. (13)C-glycine incorporation into the biomarker PLFAs for specific microbial groups (Gram positive bacteria, Gram negative bacteria, actinobacteria and fungi) was quantified using gas chromatography-combustion-stable isotope ratio mass spectrometry (GC-C-IRMS). Gram positive bacteria opportunistically utilized the freshly added glycine substrate, i.e. incorporated (13)C in all treatments, whereas fungi had minor or no glycine derived (13)C-enrichment, hence slowly reacting to a new substrate. The effects of elevated CO2 did suggest increased direct incorporation of glycine in microbial biomass, in particular in G(+) bacteria, in an ecosystem subjected to elevated CO2. Warming decreased the concentration of PLFAs in general. The FACE CO2 was (13)C-depleted (δ(13)C = 12.2‰) compared to ambient (δ(13)C = ∼-8‰), and this enabled observation of the integrated longer term responses of soil microorganisms to the FACE over one year. All together, the bacterial (and not fungal) utilization of glycine indicates substrate preference and resource partitioning in the microbial community, and therefore suggests a diversified response pattern

  1. Method for the Determination of ¹⁵N Incorporation Percentage in Labeled Peptides and Proteins.

    PubMed

    Kilpatrick, Eric L

    2016-01-01

    Use of labeled (15)N proteins and peptides as internal standards in isotope-dilution mass spectrometry for the quantification of proteins has been increasing and is now accepted as a gold standard for this analysis. As a necessary reagent in this process, stable heavy isotope-labeled internal standards must be rigorously characterized in a number of ways including identity, concentration, purity, and structure. Additionally, the degree of the incorporation of the heavy isotope is a critical feature to consider. For proteins that are (15)N labeled, the percentage of incorporation is a valid measurement used to assess the fitness-to-purpose of the material. This measurement should be objective, repeatable, and based on empirical analysis. One means of assigning this value is to compare a mass spectrum of the isotopic profile of a peptide against a series of theoretical profiles containing different enrichment rates. This comparison can be made using the Pearson product-moment correlation coefficient (r) to find the best match between the empirical and theoretical profiles. Theoretical profiles can be generated using probability multinomial analysis but are computationally intensive and require the use of computers for practical use. The method described in this chapter describes the development and use of a computer program to calculate the percentage of (15)N enrichment of a labeled internal standard. Additionally, methods will be described for the empirical determination of an isotopic profile using a variety of mass spectrometry techniques.

  2. Feasibility of Multianimal Hyperpolarized 13C MRS

    PubMed Central

    Ramirez, Marc S.; Lee, Jaehyuk; Walker, Christopher M.; Chen, Yunyun; Kingsley, Charles V.; De La Cerda, Jorge; Maldonado, Kiersten L.; Lai, Stephen Y.; Bankson, James A.

    2014-01-01

    Purpose There is great potential for real-time investigation of metabolism with MRS and hyperpolarized (HP) 13C agents. Unfortunately, HP technology has high associated costs and efficiency limitations that may constrain in vivo studies involving many animals. To improve the throughput of preclinical investigations, we evaluate the feasibility of performing HP MRS on multiple animals simultaneously. Methods Simulations helped assess the viability of a dual-coil strategy for spatially-localized multivolume MRS.A dual-mouse system was assembled and characterized based on bench- and scanner-based experiments. Enzyme phantoms mixed with HP [1-13C] pyruvate emulated real-time metabolism and offered a controlled mechanism for evaluating system performance. Finally, a normal mouse and a mouse bearing a subcutaneous xenograft of colon cancer were simultaneously scanned in vivo using an agent containing HP [1-13C] pyruvate. Results Geometric separation/rotation, active decoupling, and use of low input impedance preamplifiers permitted an encode-by-channel approach for spatially-localized MRS. A pre-calibrated shim allowed straightforward metabolite differentiation in enzyme phantom and in vivo experiments at 7 T, with performance similar to conventional acquisitions. Conclusion The initial feasibility of multi-animal HP 13C MRS was established. Throughput scales with the number of simultaneously-scanned animals, demonstrating the potential for significant improvements in study efficiency. PMID:24903532

  3. Isotopic labeling of mouse interferon by incorporation of radioactive amino acids during synthesis

    SciTech Connect

    DeMaeyer-Guignard, J.; Cachard, A.; DeMaeyer, E.

    1982-07-30

    Mouse interferon produced by C-243 cells induced with Newcastle disease virus was isotopically labeled by adding either (/sup 35/S)methionine or a /sup 14/C-labeled amino acid mixture to the culture medium. A method combining butyric acid and theophylline treatment and resulting in high interferon yields was used. Following purification by two-step affinity chromatography on poly(U) and antibody columns, the resulting material was analyzed on SDS-PAGE. The migration pattern of radioactivity and interferon coincided well and autoradiography revealed three major bands at migration distances corresponding, respectively, to 35, 28, and 22 K. Interferon represented 3.8% of all (/sup 35/S)methionine-labeled proteins and 2.6% of all /sup 14/C-amino acid-labeled proteins released into the medium.

  4. Synthesis of [1-.sup.13C]pyruvic acid], [2-.sup.13C]pyruvic acid], [3-.sup.13C]pyruvic acid] and combinations thereof

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2009-09-01

    The present invention is directed to labeled compounds, of the formulae ##STR00001## wherein C* is each independently selected from the group consisting of .sup.13C and .sup.12C with the proviso that at least one C* is .sup.13C, each hydrogen of the methylene group can independently be either hydrogen or deuterium, the methyl group includes either zero or three deuterium atoms, Q is from the group of sulfide, sulfinyl, and sulfone, Z is an aryl group from the group of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently from the group of hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group from the group of NH.sub.2, NHR and NRR' where R and R' are each independently from the group of a C.sub.1-C.sub.4 lower alkyl, a phenyl, and an alkoxy group, and the methyl group can include either zero or three deuterium atoms.

  5. Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy.

    PubMed

    Schroeder, Marie A; Atherton, Helen J; Ball, Daniel R; Cole, Mark A; Heather, Lisa C; Griffin, Julian L; Clarke, Kieran; Radda, George K; Tyler, Damian J

    2009-08-01

    The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-(13)C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of (13)C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of (13)C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-(13)C]pyruvate.

  6. High-resolution proton NMR studies of intracellular metabolites in yeast using 13C decoupling

    NASA Astrophysics Data System (ADS)

    Sillerud, Laurel O.; Alger, Jeffry R.; Shulman, Robert G.

    The resolution and specificity of 1H NMR in studies of yeast cellular metabolism were increased by feeding a 13C-labeled substrate and observing 1H difference spectra in the presence and absence of 13C decoupling fields. [2- 13C]Acetate was utilized as a respiratory substrate in an aerobic suspension of Saccharomyces cerevisiae. The broad cellular background proton resonances are removed by the technique, leaving only signals from the protons of the substrate, or its metabolites, that are coupled to 13C. Spectra of the yeast suspension after acetate feeding show the disappearance of label from the acetate pool and the subsequent appearance of 13C in glutamate C 3 and C 4 and in aspartate C 3. These results are in accord with the known fluxes of metabolites. Selective single-frequency 13C decoupling was used to provide assignments for the difference signals. The limitations on single-frequency decoupling coming from finite decoupling fields are investigated. The technique shows a potential for application in a wide variety of systems where the resolution of the 13C spectrum may be combined with the sensitivity for proton detection to observe metabolites that have been previously unobservable.

  7. {sup 13}C-{sup 1}H and {sup 13}C-{sup 13}C spin couplings in [2`-{sup 13}C]2`-deoxyribonucleosides: Correlations with molecular structure

    SciTech Connect

    Bandyopadhyay, T.; Stripe, W.A.; Carmichael, I.; Serianni, A.S.; Wu, J.

    1997-02-19

    2`-Deoxyribonucleosides (2`-deoxyadenosine (1), 2`-depoxycytidine (2), thymidine (3)) singly enriched with {sup 13}C at C2` have been prepared and used to obtain one-, two-, and three-bond {sup 13}C-{sup 1}H and {sup 13}C-{sup 13}C spin-coupling constants involving C2`. Spin couplings in 1-3 involving C1` and C2`are also compared to corresponding values in ribonucleosides in order to assess the effects of nucleoside structure and conformation on J values within the furanose ring. {sup 1}J{sub C2`,H2`R} and {sup 1}J{sub C2`,H2`S} in 1-3 and {sup 1}J{sub C2`,H2`} in ribonucleosides depend on C-H bond orientation: {sup 1}J{sub C1`,H1`} in 1-3 and in ribonucleosides exhibits a similar dependence. The latter couplings appear to be essentially unaffected by N-glycoside torsion. {sup 1}J{sub CC} values depend on the number and distribution of electronegative substituents on the C-C fragment. A modified projection curve is proposed to aid in the interpretation of {sup 2}J{sub C2`,H1`} values; the presence of N substitution at C1` caused a shift to more negative couplings relative to the O-substituted analog. In contrast, {sup 2}J{sub C1`,H2`} is essentially unaffected by the same change in the electronegative substituent at C1`. {sup 2}J{sub CC} values within the furanose ring are determined buy two coupling pathways; in one case (i.e., {sup 2}J{sub C1`,C3`}), the observed coupling is shown to be the algebraic sum of the two couplings arising from each pathway. 41 refs., 4 figs., 2 tabs.

  8. In vivo incorporation of labeled fatty acids in rat liver lipids after oral administration

    SciTech Connect

    Leyton, J.; Drury, P.J.; Crawford, M.A.

    1987-08-01

    Striking differences were found in the compartmentalization of fatty acids into liver lipid fractions. The saturated fatty acids--lauric, myristic, palmitic and stearic--were incorporated into phosphoglycerides at faster rates with increasing chain lengths, while triglyceride incorporation was almost uniform. The degree of incorporation of the unsaturated fatty acids into phosphoglycerides (structural) compared to triglyceride (storage and energy) was the converse of their oxidation rates. The incorporation of oleic, linoleic and alpha-linolenic acids was mainly into triglyceride, whereas dihomo-gamma-linolenic acid and arachidonic acid were preferentially incorporated into phosphoglycerides. The data suggest that distribution of each fatty acid is different depending on its destination for structural or energy function.

  9. A Large Metabolic Carbon Ccontribution to the δ13C Record in Marine Aragonitic Bivalve Shells

    NASA Astrophysics Data System (ADS)

    Gillikin, D. P.; Lorrain, A.; Dehairs, F.

    2006-12-01

    The stable carbon isotopic signature archived in bivalve shells was originally thought to record the δ13C of seawater dissolved inorganic carbon (δ13C-DIC). However, more recent studies have shown that the incorporation of isotopically light metabolic carbon (M) significantly affects the δ13C signal recorded in biogenic carbonates. To assess the M contribution to Mercenaria mercenaria shells collected in North Carolina, USA, we sampled seawater δ13C-DIC, tissue, hemolymph and shell δ13C. We found up to a 4‰ decrease through ontogeny in shell δ13C in a 23 year old individual. There was no correlation between shell height or age and tissue δ13C. Thus, the ontogenic decrease observed in the shell δ13C could not be attributed to changes in food sources as the animal ages leading to more negative metabolic CO2, since this would require a negative relationship between tissue δ13C and shell height. Hemolymph δ13C, on the other hand, did exhibit a negative relationship with height, but the δ13C values were more positive than expected, indicating that hemolymph may not be a good proxy of extrapallial fluid δ13C. Nevertheless, the hemolymph data indicate that respired CO2 does influence the δ13C of internal fluids and that the amount of respired CO2 is related to the age of the bivalve. The percent metabolic C incorporated into the shell (%M) was significantly higher (up to 37%) than has been found in other bivalve shells, which usually contain less than 10 %M. Attempts to use shell biometrics to predict %M could not explain more than ~60% of the observed variability. Moreover, there were large differences in the %M between different sites. Thus, the metabolic effect on shell δ13C cannot easily be accounted for to allow reliable δ13C-DIC reconstructions. However, there does seem to be a common effect of size, as all sites had indistinguishable slopes between the %M and shell height (+0.19% per mm of shell height).

  10. (13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

    PubMed

    Shestov, Alexander A; Lee, Seung-Cheol; Nath, Kavindra; Guo, Lili; Nelson, David S; Roman, Jeffrey C; Leeper, Dennis B; Wasik, Mariusz A; Blair, Ian A; Glickson, Jerry D

    2016-01-01

    We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate-malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to (13)C magnetic resonance spectroscopy ((13)C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable (13)C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of

  11. 13C MRS and LC–MS Flux Analysis of Tumor Intermediary Metabolism

    PubMed Central

    Shestov, Alexander A.; Lee, Seung-Cheol; Nath, Kavindra; Guo, Lili; Nelson, David S.; Roman, Jeffrey C.; Leeper, Dennis B.; Wasik, Mariusz A.; Blair, Ian A.; Glickson, Jerry D.

    2016-01-01

    We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate–malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to 13C magnetic resonance spectroscopy (13C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable 13C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of

  12. Specific Cellular Incorporation of a Pyrene-Labelled Cholesterol: Lipoprotein-Mediated Delivery toward Ordered Intracellular Membranes

    PubMed Central

    Gaibelet, Gérald; Azalbert, Vincent; Bertrand-Michel, Justine; Hamdi, Safouane; Collet, Xavier; Orlowski, Stéphane

    2015-01-01

    In the aim of testing tools for tracing cell trafficking of exogenous cholesterol, two fluorescent derivatives of cholesterol, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), with distinctive chemico-physical characteristics, have been compared for their cell incorporation properties, using two cell models differently handling cholesterol, with two incorporation routes. In the Caco-2 cell model, the cholesterol probes were delivered in bile salt micelles, as a model of intestinal absorption. The two probes displayed contrasting behaviors for cell uptake characteristics, cell staining, and efflux kinetics. In particular, Pyr-met-Chol cell incorporation involved SR-BI, while that of NBD-Chol appeared purely passive. In the PC-3 cell model, which overexpresses lipoprotein receptors, the cholesterol probes were delivered via the serum components, as a model of systemic delivery. We showed that Pyr-met-Chol-labelled purified LDL or HDL were able to specifically deliver Pyr-met-Chol to the PC-3 cells, while NBD-Chol incorporation was independent of lipoproteins. Observations by fluorescence microscopy evidenced that, while NBD-Chol readily stained the cytosolic lipid droplets, Pyr-met-Chol labelling led to the intense staining of intracellular structures of membranous nature, in agreement with the absence of detectable esterification of Pyr-met-Chol. A 48 h incubation of PC-3 cells with either Pyr-met-Chol-labelled LDL or HDL gave same staining patterns, mainly colocalizing with Lamp1, caveolin-1 and CD63. These data indicated convergent trafficking downwards their respective receptors, LDL-R and SR-BI, toward the cholesterol-rich internal membrane compartments, late endosomes and multivesicular bodies. Interestingly, Pyr-met-Chol staining of these structures exhibited a high excimer fluorescence emission, revealing their ordered membrane environment, and indicating that Pyr-met-Chol behaves as a fair cholesterol tracer

  13. Optoacoustic 13C-breath test analyzer

    NASA Astrophysics Data System (ADS)

    Harde, Hermann; Helmrich, Günther; Wolff, Marcus

    2010-02-01

    The composition and concentration of exhaled volatile gases reflects the physical ability of a patient. Therefore, a breath analysis allows to recognize an infectious disease in an organ or even to identify a tumor. One of the most prominent breath tests is the 13C-urea-breath test, applied to ascertain the presence of the bacterium helicobacter pylori in the stomach wall as an indication of a gastric ulcer. In this contribution we present a new optical analyzer that employs a compact and simple set-up based on photoacoustic spectroscopy. It consists of two identical photoacoustic cells containing two breath samples, one taken before and one after capturing an isotope-marked substrate, where the most common isotope 12C is replaced to a large extent by 13C. The analyzer measures simultaneously the relative CO2 isotopologue concentrations in both samples by exciting the molecules on specially selected absorption lines with a semiconductor laser operating at a wavelength of 2.744 μm. For a reliable diagnosis changes of the 13CO2 concentration of 1% in the exhaled breath have to be detected at a concentration level of this isotope in the breath of about 500 ppm.

  14. Rapid aquatic toxicity assay utilizing labeled thymidine incorporation in sea urchin embryos

    SciTech Connect

    Jackim, E.; Nacci, D.

    1984-01-01

    Aquatic toxicity was evaluated in the sea urchin embryo (Arbacea punctulata) by the inhibition of tritiated thymidine incorporation after a brief exposure to toxic chemicals. Arbacia is a useful surrogate species for assay: well-studied, easily cultured and fertile virtually year round. The simplicity and speed of this test system lends itself to screening large numbers of compounds, mixtures or water samples.

  15. Multi-Label Segmentation Propagation for Materials Science Images Incorporating Topology and Interactivity

    NASA Astrophysics Data System (ADS)

    Waggoner, Jarrell

    Segmentation propagation is the problem of transferring the segmentation of an image to a neighboring image in a sequence. This problem is of particular importance to materials science, where the accurate segmentation of a series of 2D serial-sectioned images of multiple, contiguous 3D structures has important applications. Such structures may have prior-known shape, appearance, and/or topology among the underlying structures which can be considered to improve segmentation accuracy. For example, some materials images may have structures with a specific shape or appearance in each serial section slice, which only changes minimally from slice to slice; and some materials may exhibit specific topology which constrains their structure or neighboring relations. In this work, we develop a framework for materials image segmentation that segments a variety of materials image types by repeatedly propagating a 2D segmentation from one slice to another, and we formulate each step of this propagation as an optimal labeling problem that can be efficiently solved using the graph-cut algorithm. During this propagation, we propose novel strategies to enforce the shape, appearance, and topology of the segmented structures, as well as handling local topology inconsistency. Most recent works on topology-constrained image segmentation focus on binary segmentation, where the topology is often described by the connectivity of both foreground and background. We develop a new multi-labeling approach to enforce topology in multiple-label image segmentation. In this case, we not only require each segment to be a connected region (intra-segment topology), but also require specific adjacency relations between each pair of segments (inter-segment topology). Finally, we integrate an interactive approach into the proposed framework that improves the segmentation by allowing minimal and simplistic human annotations. We justify the effectiveness of the proposed framework by testing it on various 3

  16. A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

    PubMed Central

    2011-01-01

    Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits against experimental data

  17. 13C editing of glutamate in human brain using J-refocused coherence transfer spectroscopy at 4.1 T.

    PubMed

    Pan, J W; Mason, G F; Vaughan, J T; Chu, W J; Zhang, Y; Hetherington, H P

    1997-03-01

    The method of single quantum 13C editing is analyzed and implemented with water suppressed J-refocused coherence transfer spectroscopy. Analysis of the 13C inversion pulse demonstrates that it is optimally placed into the second echo of the J-refocused sequence. We have used this method to acquire 13C-edited spectra of glutamate from phantoms and in vivo. The turnover of 13C4-labeled glutamate in human brain in vivo was observed in parasagittal gray matter using a volume head coil at 4.1 T with a time resolution of 5.3 min.

  18. Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis

    PubMed Central

    2014-01-01

    This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields — the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route — in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories. PMID:24642094

  19. Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis.

    PubMed

    Varman, Arul M; He, Lian; You, Le; Hollinshead, Whitney; Tang, Yinjie J

    2014-03-19

    This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields - the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route - in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories.

  20. Dynamic or inert metabolism? Turnover of N-acetyl aspartate and glutathione from D-[1-13C]glucose in the rat brain in vivo

    PubMed Central

    Choi, In-Young; Gruetter, Rolf

    2006-01-01

    The rate of 13C-label incorporation into both aspartyl (NAA C3) and acetyl (NAA C6) groups of N-acetyl aspartate (NAA) was simultaneously measured in the rat brain in vivo for up to 19 h of [1-13C]glucose infusion (n = 8). Label incorporation was detected in NAA C6 approximately 1.5 h earlier than in NAA C3 because of the delayed labeling of the precursor of NAA C3, aspartate, compared to that of NAA C6, glucose. The time courses of NAA were fitted using a mathematical model assuming synthesis of NAA in one kinetic compartment with the respective precursor pools of aspartate and acetyl coenzyme A (acetyl-CoA). The turnover rates of NAA C6 and C3 were 0.7 ± 0.1 and 0.6 ± 0.1 μmol/(g h) with the time constants 14 ± 2 and 13 ± 2 h, respectively, with an estimated pool size of 8 μmol/g. The results suggest that complete label turnover of NAA from glucose occurs in approximately 70 h. Several hours after starting the glucose infusion, label incorporation into glutathione (GSH) was also detected. The turnover rate of GSH was 0.06 ± 0.02 μmol/(g h) with a time constant of 13 ± 2 h. The estimated pool size of GSH was 0.8 μmol/g, comparable to the cortical glutathione concentration. We conclude that NAA and GSH are completely turned over and that the metabolism is extremely slow (< 0.05% of the glucose metabolic rate). PMID:15525331

  1. 13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea.

    PubMed

    Glaubitz, Sabine; Lueders, Tillmann; Abraham, Wolf-Rainer; Jost, Günter; Jürgens, Klaus; Labrenz, Matthias

    2009-02-01

    Marine pelagic redoxclines are zones of high dark CO(2) fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO(2) fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO(2) incorporation in (13)C-bicarbonate pulse experiments. The incorporation of (13)C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in (13)C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO(2)-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the (13)C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO(2)-fixing Proteobacteria within this habitat.

  2. 13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea.

    PubMed

    Glaubitz, Sabine; Lueders, Tillmann; Abraham, Wolf-Rainer; Jost, Günter; Jürgens, Klaus; Labrenz, Matthias

    2009-02-01

    Marine pelagic redoxclines are zones of high dark CO(2) fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO(2) fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO(2) incorporation in (13)C-bicarbonate pulse experiments. The incorporation of (13)C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in (13)C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO(2)-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the (13)C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO(2)-fixing Proteobacteria within this habitat. PMID:18793316

  3. Multi-objective experimental design for (13)C-based metabolic flux analysis.

    PubMed

    Bouvin, Jeroen; Cajot, Simon; D'Huys, Pieter-Jan; Ampofo-Asiama, Jerry; Anné, Jozef; Van Impe, Jan; Geeraerd, Annemie; Bernaerts, Kristel

    2015-10-01

    (13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi

  4. Incorporation of isotope from specifically labeled glucose into alginates of Pseudomonas aeruginosa and Azotobacter vinelandii.

    PubMed Central

    Lynn, A R; Sokatch, J R

    1984-01-01

    The incorporation of isotope from [6-14C]glucose into alginate by both Pseudomonas aeruginosa and Azotobacter vinelandii was 10-fold greater than that from either [1-14C]- or [2-14C]glucose, indicating preferential utilization of the bottom half of the glucose molecule for alginate synthesis. These data strongly suggest that the Entner - Doudoroff pathway plays a major role in alginate synthesis in both P. aeruginosa and A. vinelandii. PMID:6427189

  5. Bacteria and Fungi Respond Differently to Multifactorial Climate Change in a Temperate Heathland, Traced with 13C-Glycine and FACE CO2

    PubMed Central

    Andresen, Louise C.; Dungait, Jennifer A. J.; Bol, Roland; Selsted, Merete B.; Ambus, Per; Michelsen, Anders

    2014-01-01

    It is vital to understand responses of soil microorganisms to predicted climate changes, as these directly control soil carbon (C) dynamics. The rate of turnover of soil organic carbon is mediated by soil microorganisms whose activity may be affected by climate change. After one year of multifactorial climate change treatments, at an undisturbed temperate heathland, soil microbial community dynamics were investigated by injection of a very small concentration (5.12 µg C g−1 soil) of 13C-labeled glycine (13C2, 99 atom %) to soils in situ. Plots were treated with elevated temperature (+1°C, T), summer drought (D) and elevated atmospheric carbon dioxide (510 ppm [CO2]), as well as combined treatments (TD, TCO2, DCO2 and TDCO2). The 13C enrichment of respired CO2 and of phospholipid fatty acids (PLFAs) was determined after 24 h. 13C-glycine incorporation into the biomarker PLFAs for specific microbial groups (Gram positive bacteria, Gram negative bacteria, actinobacteria and fungi) was quantified using gas chromatography-combustion-stable isotope ratio mass spectrometry (GC-C-IRMS). Gram positive bacteria opportunistically utilized the freshly added glycine substrate, i.e. incorporated 13C in all treatments, whereas fungi had minor or no glycine derived 13C-enrichment, hence slowly reacting to a new substrate. The effects of elevated CO2 did suggest increased direct incorporation of glycine in microbial biomass, in particular in G+ bacteria, in an ecosystem subjected to elevated CO2. Warming decreased the concentration of PLFAs in general. The FACE CO2 was 13C-depleted (δ13C = 12.2‰) compared to ambient (δ13C = ∼−8‰), and this enabled observation of the integrated longer term responses of soil microorganisms to the FACE over one year. All together, the bacterial (and not fungal) utilization of glycine indicates substrate preference and resource partitioning in the microbial community, and therefore suggests a diversified response pattern to future

  6. Fractional enrichment of proteins using [2-(13)C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity.

    PubMed

    Ahlner, Alexandra; Andresen, Cecilia; Khan, Shahid N; Kay, Lewis E; Lundström, Patrik

    2015-07-01

    A selective isotope labeling scheme based on the utilization of [2-(13)C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state (13)Cα chemical shifts using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-(13)C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state (13)Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s(-1), despite the small fraction of (13)Cα-(13)Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using (13)Cα spin probes.

  7. A method for simultaneous echo planar imaging of hyperpolarized 13C pyruvate and 13C lactate

    NASA Astrophysics Data System (ADS)

    Reed, Galen D.; Larson, Peder E. Z.; von Morze, Cornelius; Bok, Robert; Lustig, Michael; Kerr, Adam B.; Pauly, John M.; Kurhanewicz, John; Vigneron, Daniel B.

    2012-04-01

    A rapid echo planar imaging sequence for dynamic imaging of [1-13C] lactate and [1-13C] pyruvate simultaneously was developed. Frequency-based separation of these metabolites was achieved by spatial shifting in the phase-encoded direction with the appropriate choice of echo spacing. Suppression of the pyruvate-hydrate and alanine resonances is achieved through an optimized spectral-spatial RF waveform. Signal sampling efficiency as a function of pyruvate and lactate excitation angle was simulated using two site exchange models. Dynamic imaging is demonstrated in a transgenic mouse model, and phantom validations of the RF pulse frequency selectivity were performed.

  8. An overview of methods using 13C for improved compound identification in metabolomics and natural products

    PubMed Central

    Clendinen, Chaevien S.; Stupp, Gregory S.; Ajredini, Ramadan; Lee-McMullen, Brittany; Beecher, Chris; Edison, Arthur S.

    2015-01-01

    Compound identification is a major bottleneck in metabolomics studies. In nuclear magnetic resonance (NMR) investigations, resonance overlap often hinders unambiguous database matching or de novo compound identification. In liquid chromatography-mass spectrometry (LC-MS), discriminating between biological signals and background artifacts and reliable determination of molecular formulae are not always straightforward. We have designed and implemented several NMR and LC-MS approaches that utilize 13C, either enriched or at natural abundance, in metabolomics applications. For LC-MS applications, we describe a technique called isotopic ratio outlier analysis (IROA), which utilizes samples that are isotopically labeled with 5% (test) and 95% (control) 13C. This labeling strategy leads to characteristic isotopic patterns that allow the differentiation of biological signals from artifacts and yield the exact number of carbons, significantly reducing possible molecular formulae. The relative abundance between the test and control samples for every IROA feature can be determined simply by integrating the peaks that arise from the 5 and 95% channels. For NMR applications, we describe two 13C-based approaches. For samples at natural abundance, we have developed a workflow to obtain 13C–13C and 13C–1H statistical correlations using 1D 13C and 1H NMR spectra. For samples that can be isotopically labeled, we describe another NMR approach to obtain direct 13C–13C spectroscopic correlations. These methods both provide extensive information about the carbon framework of compounds in the mixture for either database matching or de novo compound identification. We also discuss strategies in which 13C NMR can be used to identify unknown compounds from IROA experiments. By combining technologies with the same samples, we can identify important biomarkers and corresponding metabolites of interest. PMID:26379677

  9. Confirmation of Fructans biosynthesized in vitro from [1-13C]glucose in asparagus tissues using MALDI-TOF MS and ESI-MS.

    PubMed

    Suzuki, Takashi; Maeda, Tomoo; Grant, Suzanne; Grant, Gordon; Sporns, Peter

    2013-05-15

    Accumulation of Fructans was confirmed in asparagus tissues that had been cultured for 2 days on media supplemented with glucose. It is very common that Fructans are biosynthesized from sucrose. We hypothesized however that Fructans could also be biosynthesized from glucose. Stem tissues of in vitro-cultured asparagus were subcultured for 72 h on a medium containing 0.5M of [1-(13)C]glucose. A medium containing 0.5M of normal ((12)C) glucose was used as control. Carbohydrates were extracted from the tissues and analyzed using HPLC, MALDI-TOF MS and ESI-MS. HPLC results indicated that the accumulation of short-chain Fructans was similar in both (13)C-labelled and control samples. Short-chain Fructans of DP=3-7 were detected using MALDI-TOF MS. The molecular mass of each oligomer in the (13)C-labelled sample was higher than the mass of the natural sample by 1 m/z unit per sugar moiety. The results of ESI-MS on the HPLC fractions of neokestose and 1-kestose showed that these oligomers (DP=3) were biosynthesized from exogenous glucose added to the medium. We conclude that not only exogenous sucrose but glucose can induce Fructan biosynthesis; fructans of both inulin type and inulin neoseries are also biosynthesized from glucose accumulated in asparagus tissues; the glucose molecules (or its metabolic products) were incorporated into Fructans as structural monomers.

  10. Effects of fasting on serial measurements of hyperpolarized [1-(13) C]pyruvate metabolism in tumors.

    PubMed

    Serrao, Eva M; Rodrigues, Tiago B; Gallagher, Ferdia A; Kettunen, Mikko I; Kennedy, Brett W C; Vowler, Sarah L; Burling, Keith A; Brindle, Kevin M

    2016-08-01

    Imaging of the metabolism of hyperpolarized [1-(13) C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of (13) C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non-fasted animals. The fasted state showed lower intra-individual variability, although the [1-(13) C]lactate/[1-(13) C]pyruvate signal ratio was significantly greater in fasted than in non-fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of (13) C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:27309986

  11. Effects of fasting on serial measurements of hyperpolarized [1‐13C]pyruvate metabolism in tumors

    PubMed Central

    Serrao, Eva M.; Rodrigues, Tiago B.; Gallagher, Ferdia A.; Kettunen, Mikko I.; Kennedy, Brett W. C.; Vowler, Sarah L.; Burling, Keith A.

    2016-01-01

    Imaging of the metabolism of hyperpolarized [1‐13C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of 13C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non‐fasted animals. The fasted state showed lower intra‐individual variability, although the [1‐13C]lactate/[1‐13C]pyruvate signal ratio was significantly greater in fasted than in non‐fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of 13C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:27309986

  12. Strength and limits using 13C phospholipid fatty acid analysis in soil ecology

    NASA Astrophysics Data System (ADS)

    Watzinger, Andrea

    2016-04-01

    This presentation on microbial phospholipid biomarkers, their isotope analysis and their ability to reveal soil functions summarizes experiences gained by the author for more than 10 years. The amount and composition of phospholipid fatty acids (PLFAs) measured in environmental samples strongly depend on the methodology. To achieve comparable results the extraction, separation and methylation method must be kept constant. PLFAs patterns are sensitive to microbial community shifts even though the taxonomic resolution of PLFAs is low. The possibility to easily link lipid biomarkers with stable isotope techniques is identified as a major advantage when addressing soil functions. Measurement of PLFA isotopic ratios is sensitive and enables detecting isotopic fractionation. The difference between the carbon isotopic ratio of single PLFAs and their substrate (δ13C) can vary between -6 and +11‰. This difference derives from the fractionation during biosynthesis and from substrate inhomogeneity. Consequently, natural abundance studies are restricted to quantifying substrate uptake of the total microbial biomass. In contrast, artificial labelling enables quantifying carbon uptake into single PLFAs, but labelling success depends on homogeneous and undisturbed label application. Current developments in microbial ecology (e.g. 13C and 15N proteomics) and isotope techniques (online monitoring of CO2 isotope ratios) will likely improve soil functional interpretations in the future. 13C PLFA analysis will continue to contribute because it is affordable, sensitive and allows frequent sampling combined with the use of small amounts of 13C label.

  13. Calculation of total meal d13C from individual food d13C.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Variations in the isotopic signature of carbon in biological samples can be used to distinguish dietary patterns and monitor shifts in metabolism. But for these variations to have meaning, the isotopic signature of the diet must be known. We sought to determine if knowledge of the 13C isotopic abund...

  14. The delta 13C record of Devonian to Permian carbonates

    NASA Astrophysics Data System (ADS)

    Buggisch, W.

    2003-04-01

    A δ13Ccarb curve will be presented for samples spanning the time interval from the Silurian/Devonian to the Permian/Triassic boundary. Reliable data are usually based on analyses of brachiopod shells. Because of the huge reservoir of carbon in carbonates, also whole rock samples are suitable for stable carbon isotope analyses if they are not altered by meteoric water or by incorporation of re-oxidized organic carbon during diagenesis. There are several possibilities to test the quality of the data: (1) comparison of the δ13C record of whole rock samples with samples from brachiopod shells, (2) with the organic record, (3) analyses of the same time interval in different sections. If the same pattern of isotope data is observed in separa-ted palaeogeographic settings, it is probably caused by a change in the global carbon reservoir. Reliable δ13C data will be presented for the Devonian, Mississippian and Middle to Late Permian. During Pennsylvanian and Early Permian most carbonates were affected by meteoric diagenesis due to the large glacio-eustatic sea level changes of the Permo-Carboniferous glaciation. Long term variations (mean values for 10 Ma) are known from literature. Devonian δ13C values are about 0 to 2 ppm (V-PDB) they increase up to 5 to 6 ppm during the Mississippian - Pennsylvanian transition and drop sharply at the Permian Triassic boundary. The Devonian - Carboniferous trend is probably at least partly due to the evolution of land plants. Short term variations in the range of 0.1 to 1 Ma modify the long term trend significantly. Large positive excursions of δ13C up to 5 or 6 ppm are known from the Silurian - Devonian boundary and during the Middle Tournaisian of Laurentia and Europe. Many positive excursions of a magnitude of 2 to 3 ppm are observed, some are verified worldwide as for instance at the Frasnian - Famennian boundary which coincides with one of the largest extinction events in earth history. Short time variations in the isotopic

  15. Incorporation mechanisms of a branched nonylphenol isomer in soil-derived organo-clay complexes during a 180-day experiment.

    PubMed

    Riefer, Patrick; Klausmeyer, Timm; Adams, Alina; Schmidt, Burkhard; Schäffer, Andreas; Schwarzbauer, Jan

    2013-07-01

    The incorporation process of a defined (13)C- and (14)C-labeled nonylphenol isomer (4-(3,5-dimethylhept-3-yl)phenol) into soil-derived organo-clay complexes was investigated. Isolated organo-clay complexes were separated into humic subfractions. Noninvasive ((13)C-CP/MAS NMR) and invasive methods (sequential chemical degradation, pyrolysis) were applied to obtain detailed information about the mode of incorporation, chemical structure, and change of the incorporation character of nonextractable residues in course of incubation. (13)C-CP/MAS NMR measurements of humic acids revealed an increasing incorporation of phenolic compounds during the experimental time which was referred to residues of the introduced (13)C-labeled NP isomer. Detailed investigations by means of sequential chemical degradation indicated a predominant incorporation of nonextractable NP isomer residues via reversible ester (amide) bonds. In course of time, the amount of releasable compounds decreased, pointing to altering processes which affected the mode of incorporation. BBr3-treatment, RuO4 oxidation, and thermochemolysis released only low portions of nonextractable radioactivity giving evidence of strongly incorporated residues. With the comprehensive application of complementary methods (e.g., humic matter fractionation, (13)C-CP/MAS NMR, sequential chemical degradation) it was possible to provide a comparatively detailed insight into the incorporation behavior of the applied NP isomer. PMID:23713749

  16. Incorporation mechanisms of a branched nonylphenol isomer in soil-derived organo-clay complexes during a 180-day experiment.

    PubMed

    Riefer, Patrick; Klausmeyer, Timm; Adams, Alina; Schmidt, Burkhard; Schäffer, Andreas; Schwarzbauer, Jan

    2013-07-01

    The incorporation process of a defined (13)C- and (14)C-labeled nonylphenol isomer (4-(3,5-dimethylhept-3-yl)phenol) into soil-derived organo-clay complexes was investigated. Isolated organo-clay complexes were separated into humic subfractions. Noninvasive ((13)C-CP/MAS NMR) and invasive methods (sequential chemical degradation, pyrolysis) were applied to obtain detailed information about the mode of incorporation, chemical structure, and change of the incorporation character of nonextractable residues in course of incubation. (13)C-CP/MAS NMR measurements of humic acids revealed an increasing incorporation of phenolic compounds during the experimental time which was referred to residues of the introduced (13)C-labeled NP isomer. Detailed investigations by means of sequential chemical degradation indicated a predominant incorporation of nonextractable NP isomer residues via reversible ester (amide) bonds. In course of time, the amount of releasable compounds decreased, pointing to altering processes which affected the mode of incorporation. BBr3-treatment, RuO4 oxidation, and thermochemolysis released only low portions of nonextractable radioactivity giving evidence of strongly incorporated residues. With the comprehensive application of complementary methods (e.g., humic matter fractionation, (13)C-CP/MAS NMR, sequential chemical degradation) it was possible to provide a comparatively detailed insight into the incorporation behavior of the applied NP isomer.

  17. Σ production from targets of ^4He and ^13C

    NASA Astrophysics Data System (ADS)

    Chrien, R. E.

    1996-10-01

    One of the abiding issues in hypernuclear research has been the question of the formation of nuclear bound states incorporating the Σ-hyperon. The recent increases in beam intensity at the Brookhaven AGS have enabled us to obtain a high statistics study on the production of Σ-hyperons on a ^4He target. Earlier research using stopped kaons at KEK indicated the presence of structure in the (K^-,π^-) reaction, and led to the postulate of a Σ bound state. That structure has now been definitely confirmed in the in-flight kaon experiment at the LESB2 beam line and Moby-Dick spectrometer. An improved measurement of the binding energy of the presumed state will be reported, together with a production cross section. In addition, both (K^-,π^-) and (K^-,π^+) reactions on ^13C have been studied and will be compared to similar measurements on ^9Be.

  18. Growth activity in human septal cartilage: age-dependent incorporation of labeled sulfate in different anatomic locations

    SciTech Connect

    Vetter, U.; Pirsig, W.; Heinze, E.

    1983-02-01

    Growth activity in different areas of human septal cartilage was measured by the in vitro incorporation of /sup 35/S-labeled NaSO/sub 4/ into chondroitin sulfate. Septal cartilage without perichondrium was obtained during rhinoplasty from 36 patients aged 6 to 35 years. It could be shown that the anterior free end of the septum displays high growth activity in all age groups. The supra-premaxillary area displayed its highest growth activity during prepuberty, showing thereafter a continuous decline during puberty and adulthood. A similar age-dependent pattern in growth activity was found in the caudal prolongation of the septal cartilage. No age-dependent variations could be detected in the posterior area of the septal cartilage.

  19. Clinical NOE 13C MRS for neuropsychiatric disorders of the frontal lobe

    NASA Astrophysics Data System (ADS)

    Sailasuta, Napapon; Robertson, Larry W.; Harris, Kent C.; Gropman, Andrea L.; Allen, Peter S.; Ross, Brian D.

    2008-12-01

    In this communication, a scheme is described whereby in vivo 13C MRS can safely be performed in the frontal lobe, a human brain region hitherto precluded on grounds of SAR, but important in being the seat of impaired cognitive function in many neuropsychiatric and developmental disorders. By combining two well known features of 13C NMR—the use of low power NOE and the focus on 13C carbon atoms which are only minimally coupled to protons, we are able to overcome the obstacle of SAR and develop means of monitoring the 13C fluxes of critically important metabolic pathways in frontal brain structures of normal volunteers and patients. Using a combination of low-power WALTZ decoupling, variants of random noise for nuclear overhauser effect enhancement it was possible to reduce power deposition to 20% of the advised maximum specific absorption rate (SAR). In model solutions 13C signal enhancement achieved with this scheme were comparable to that obtained with WALTZ-4. In human brain, the low power procedure effectively determined glutamine, glutamate and bicarbonate in the posterior parietal brain after [1- 13C] glucose infusion. The same 13C enriched metabolites were defined in frontal brain of human volunteers after administration of [1- 13C] acetate, a recognized probe of glial metabolism. Time courses of incorporation of 13C into cerebral glutamate, glutamine and bicarbonate were constructed. The results suggest efficacy for measurement of in vivo cerebral metabolic rates of the glutamate-glutamine and tricarboxylic acid cycles in 20 min MR scans in previously inaccessible brain regions in humans at 1.5T. We predict these will be clinically useful biomarkers in many human neuropsychiatric and genetic conditions.

  20. Topological Constraints on Chain-Folding Structure of Semicrystalline Polymer as Studied by 13C-13C Double Quantum NMR

    NASA Astrophysics Data System (ADS)

    Hong, Youlee; Miyoshi, Toshikazu

    Chain-folding process is a prominent feature of long polymer chains during crystallization. Over the last half century, much effort has been paid to reveal the chain trajectory. Even though various chain-folding models as well as theories of crystallization at molecule levels have been proposed, they could be not reconciled due to the limited experimental evidences. Recent development of double quantum NMR with selective isotope labeling identified the chain-trajectory of 13C labeled isotactic poly(1-butene). The systematic experiments covered a wide range of parameters, i.e. kinetics, concentration, and molecular weight (Mw) . It was demonstrated that i) adjacent re-entry site was invariant as a function of crystallization temperature (Tc) , concentration, andMw, ii) long-range order of adjacent re-entry sequence is independence of kinetics at a given concentration while it decreased with increasing the polymer concentration at a given Tc due to the increased interruption between the chains, and iii) high Mw chains led to the multilayer folded structures in single crystals, but the melt state induced the identical short adjacent sequences of long and short polymer over a wide range of Tc due to the entanglements. The behaviors indicated that the topological restriction plays significant roles in the chain-folding process rather than the kinetics. The proposed framework to control the chain-folding structure presents a new perspective into the chain organization by either the intra- or inter-chain interaction. National Science Foundation Grants DMR-1105829 and 1408855.

  1. Neuroprotective effects of caffeine in MPTP model of Parkinson's disease: A (13)C NMR study.

    PubMed

    Bagga, Puneet; Chugani, Anup N; Patel, Anant B

    2016-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of nigrostriatal dopaminergic neurons with an accompanying neuroinflammation leading to loss of dopamine in the basal ganglia. Caffeine, a well-known A2A receptor antagonist is reported to slow down the neuroinflammation caused by activated microglia and reduce the extracellular glutamate in the brain. In this study, we have evaluated the neuroprotective effect of caffeine in the MPTP model of PD by monitoring the region specific cerebral energy metabolism. Adult C57BL6 mice were treated with caffeine (30 mg/kg, i.p.) 30 min prior to MPTP (25 mg/kg, i.p.) administration for 8 days. The paw grip strength of mice was assessed in order to evaluate the motor function after various treatments. For metabolic studies, mice were infused with [1,6-(13)C2]glucose, and (13)C labeling of amino acids was monitored using ex vivo(1)H-[(13)C]-NMR spectroscopy. The paw grip strength was found to be reduced following the MPTP treatment. The caffeine pretreatment showed significant protection against the reduction of paw grip strength in MPTP treated mice. The levels of GABA and myo-inositol were found to be elevated in the striatum of MPTP treated mice. The (13)C labeling of GluC4, GABAC2 and GlnC4 from [1,6-(13)C2]glucose was decreased in the cerebral cortex, striatum, olfactory bulb, thalamus and cerebellum suggesting impaired glutamatergic and GABAergic neuronal activity and neurotransmission of the MPTP treated mice. Most interestingly, the pretreatment of caffeine maintained the (13)C labeling of amino acids to the control values in cortical, olfactory bulb and cerebellum regions while it partially retained in striatal and thalamic regions in MPTP treated mice. The pretreatment of caffeine provides a partial neuro-protection against severe striatal degeneration in the MPTP model of PD.

  2. Dynamic nuclear polarization-enhanced 1H-13C double resonance NMR in static samples below 20 K

    NASA Astrophysics Data System (ADS)

    Potapov, Alexey; Thurber, Kent R.; Yau, Wai-Ming; Tycko, Robert

    2012-08-01

    We demonstrate the feasibility of one-dimensional and two-dimensional 1H-13C double resonance NMR experiments with dynamic nuclear polarization (DNP) at 9.4 T and temperatures below 20 K, including both 1H-13C cross-polarization and 1H decoupling, and discuss the effects of polarizing agent type, polarizing agent concentration, temperature, and solvent deuteration. We describe a two-channel low-temperature DNP/NMR probe, capable of carrying the radio-frequency power load required for 1H-13C cross-polarization and high-power proton decoupling. Experiments at 8 K and 16 K reveal a significant T2 relaxation of 13C, induced by electron spin flips. Carr-Purcell experiments and numerical simulations of Carr-Purcell dephasing curves allow us to determine the effective correlation time of electron flips under our experimental conditions. The dependence of the DNP signal enhancement on electron spin concentration shows a maximum near 80 mM. Although no significant difference in the absolute DNP enhancements for triradical (DOTOPA-TEMPO) and biradical (TOTAPOL) dopants was found, the triradical produced greater DNP build-up rates, which are advantageous for DNP experiments. Additionally the feasibility of structural measurements on 13C-labeled biomolecules was demonstrated with a two-dimensional 13C-13C exchange spectrum of selectively 13C-labeled β-amyloid fibrils.

  3. IsoDesign: a software for optimizing the design of 13C-metabolic flux analysis experiments.

    PubMed

    Millard, Pierre; Sokol, Serguei; Letisse, Fabien; Portais, Jean-Charles

    2014-01-01

    The growing demand for (13) C-metabolic flux analysis ((13) C-MFA) in the field of metabolic engineering and systems biology is driving the need to rationalize expensive and time-consuming (13) C-labeling experiments. Experimental design is a key step in improving both the number of fluxes that can be calculated from a set of isotopic data and the precision of flux values. We present IsoDesign, a software that enables these parameters to be maximized by optimizing the isotopic composition of the label input. It can be applied to (13) C-MFA investigations using a broad panel of analytical tools (MS, MS/MS, (1) H NMR, (13) C NMR, etc.) individually or in combination. It includes a visualization module to intuitively select the optimal label input depending on the biological question to be addressed. Applications of IsoDesign are described, with an example of the entire (13) C-MFA workflow from the experimental design to the flux map including important practical considerations. IsoDesign makes the experimental design of (13) C-MFA experiments more accessible to a wider biological community. IsoDesign is distributed under an open source license at http://metasys.insa-toulouse.fr/software/isodes/

  4. 13C isotopic fractionation during biodegradation of agricultural wastes.

    PubMed

    Chalk, Phillip M; Inácio, Caio T; Urquiaga, Segundo; Chen, Deli

    2015-01-01

    Significant differences in δ(13)C signatures occur within and between plant tissues and their constituent biochemical entities, and also within and between heterotrophic bacteria and fungi and their metabolic products. Furthermore, (13)C isotopic fractionation occurs during the biodegradation of organic molecules as seen in the substrate, respired CO(2) and the microbial biomass, which could be related to substrate composition and/or microbial metabolism. The (13)C isotopic fractionation observed during the decomposition of a single defined C substrate appears to be due to the intra-molecular heterogeneity in (13)C in the substrate and to (13)C isotopic fractionation during microbial metabolism. Very limited data suggest that the latter may be quantitatively more important than the former. Studies with defined fungi in culture media have highlighted the complexities associated with the interpretation of the observed patterns of (13)C isotopic fractionation when a single defined C source is added to the culture medium which itself contains one or more C sources. Techniques involving (13)C enrichment or paired treatments involving an equivalent C(3)- and C(4)-derived substrate have been devised to overcome the problem of background C in the culture medium and (13)C isotopic fractionation during metabolism. Studies with complex substrates have shown an initial (13)C depletion phase in respired CO(2) followed by a (13)C enrichment phase which may or may not be followed by another (13)C depletion phase. Basic studies involving an integrated approach are required to gain a new insight into (13)C isotopic fractionation during organic residue decomposition, by simultaneous measurements of δ(13)C in all C moieties. New analytical tools to measure real-time changes in δ(13)CO(2) and the intra-molecular δ(13)C distribution within plant biochemical entities offer new opportunities for unravelling the complex interactions between substrate and microbial metabolism with

  5. (13)C-Breath testing in animals: theory, applications, and future directions.

    PubMed

    McCue, Marshall D; Welch, Kenneth C

    2016-04-01

    The carbon isotope values in the exhaled breath of an animal mirror the carbon isotope values of the metabolic fuels being oxidized. The measurement of stable carbon isotopes in carbon dioxide is called (13)C-breath testing and offers a minimally invasive method to study substrate oxidation in vivo. (13)C-breath testing has been broadly used to study human exercise, nutrition, and pathologies since the 1970s. Owing to reduced use of radioactive isotopes and the increased convenience and affordability of (13)C-analyzers, the past decade has witnessed a sharp increase in the use of breath testing throughout comparative physiology--especially to answer questions about how and when animals oxidize particular nutrients. Here, we review the practical aspects of (13)C-breath testing and identify the strengths and weaknesses of different methodological approaches including the use of natural abundance versus artificially-enriched (13)C tracers. We critically compare the information that can be obtained using different experimental protocols such as diet-switching versus fuel-switching. We also discuss several factors that should be considered when designing breath testing experiments including extrinsic versus intrinsic (13)C-labelling and different approaches to model nutrient oxidation. We use case studies to highlight the myriad applications of (13)C-breath testing in basic and clinical human studies as well as comparative studies of fuel use, energetics, and carbon turnover in multiple vertebrate and invertebrate groups. Lastly, we call for increased and rigorous use of (13)C-breath testing to explore a variety of new research areas and potentially answer long standing questions related to thermobiology, locomotion, and nutrition. PMID:26660654

  6. (13)C-Breath testing in animals: theory, applications, and future directions.

    PubMed

    McCue, Marshall D; Welch, Kenneth C

    2016-04-01

    The carbon isotope values in the exhaled breath of an animal mirror the carbon isotope values of the metabolic fuels being oxidized. The measurement of stable carbon isotopes in carbon dioxide is called (13)C-breath testing and offers a minimally invasive method to study substrate oxidation in vivo. (13)C-breath testing has been broadly used to study human exercise, nutrition, and pathologies since the 1970s. Owing to reduced use of radioactive isotopes and the increased convenience and affordability of (13)C-analyzers, the past decade has witnessed a sharp increase in the use of breath testing throughout comparative physiology--especially to answer questions about how and when animals oxidize particular nutrients. Here, we review the practical aspects of (13)C-breath testing and identify the strengths and weaknesses of different methodological approaches including the use of natural abundance versus artificially-enriched (13)C tracers. We critically compare the information that can be obtained using different experimental protocols such as diet-switching versus fuel-switching. We also discuss several factors that should be considered when designing breath testing experiments including extrinsic versus intrinsic (13)C-labelling and different approaches to model nutrient oxidation. We use case studies to highlight the myriad applications of (13)C-breath testing in basic and clinical human studies as well as comparative studies of fuel use, energetics, and carbon turnover in multiple vertebrate and invertebrate groups. Lastly, we call for increased and rigorous use of (13)C-breath testing to explore a variety of new research areas and potentially answer long standing questions related to thermobiology, locomotion, and nutrition.

  7. Radioautography of rat incisor dentin as a continuous record of the incorporation of a single dose of /sup 3/H-labeled proline and tyrosine

    SciTech Connect

    Josephsen, K.; Warshawsky, H.

    1982-05-01

    After injection of labeled precursors such as /sup 3/H-proline or /sup 3/H-tyrosine into rats, the incisor dentin contains a continuous and stable record of precursor incorporation into labeled proteins. This record was visualized and quantitated with radioautography in order to evaluate the quantitative changes in enamel where newly secreted proteins randomize with older proteins and both are eventually lost. Up to 4 hours after injection, the pulse-dose was incorporated as a highly labeled band of predentin. The band was entirely within calcified dentin at 2 days and was further removed from new predentin by 4 and 8 days. Dentin which formed proximal to the heavily labeled band contained an amount of radioactivity reflecting the level of labeled precursor available at that time. A standardizing factor for experimental error was obtained by quantitating the reaction in the heavily labeled band, and a post-pulse incorporation factor was determined from the amount of radioactivity added per day as weakly labeled dentin. The variation within the heavily labeled band was assumed to reflect experimental error. The number of grains in the bands were averaged from 4 hours to 8 days to give the standardizing factor. This was multiplied by the ratio of enamel to dentin counts in the same section to obtain a corrected enamel count. With proline it amounted to 5% increase per day from 1 to 4 days and 2.5% per day from 4 to 8 days after injection. In addition, with /sup 3/H-proline the incorporation into predentin increased from 30 minutes to 4 hours. With tyrosine, the counts increased from 30 minutes to 1 hour, but decreased by nearly one third from 1 to 4 hours. This was interpreted as a loss of short-lived matrix proteins including procollagen peptides produced during conversion from procollagen to tropocollagen in the predentin.

  8. 13C magnetic resonance spectroscopy measurements with hyperpolarized [1‐13C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo

    PubMed Central

    Dzien, Piotr; Tee, Sui‐Seng; Kettunen, Mikko I.; Lyons, Scott K.; Larkin, Timothy J.; Timm, Kerstin N.; Hu, De‐En; Wright, Alan; Rodrigues, Tiago B.; Serrao, Eva M.; Marco‐Rius, Irene; Mannion, Elizabeth; D'Santos, Paula; Kennedy, Brett W. C.

    2015-01-01

    Purpose Dissolution dynamic nuclear polarization can increase the sensitivity of the 13C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize 13C‐labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. Methods Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using 13C MRS measurements of the conversion of hyperpolarized [1‐13C] pyruvate to H13 CO3–. Results Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two‐fold increase in the H13 CO3–/[1‐13C] pyruvate signal ratio following intravenous injection of hyperpolarized [1‐13C] pyruvate. Conclusion We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized 13C MRS. Magn Reson Med 76:391–401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:26388418

  9. (13)C metabolic flux analysis of the extremely thermophilic, fast growing, xylose-utilizing Geobacillus strain LC300.

    PubMed

    Cordova, Lauren T; Antoniewicz, Maciek R

    2016-01-01

    Thermophiles are increasingly used as versatile hosts in the biotechnology industry. One of the key advantages of thermophiles is the potential to achieve high rates of feedstock conversion at elevated temperatures. The recently isolated Geobacillus strain LC300 grows extremely fast on xylose, with a doubling time of less than 30 min. In the accompanying paper, the genome of Geobacillus LC300 was sequenced and annotated. In this work, we have experimentally validated the metabolic network model using parallel (13)C-labeling experiments and applied (13)C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], and [5-(13)C]xylose, was used for the first time. Isotopic labeling of biomass amino acids was measured by gas chromatography mass spectrometry (GC-MS). Isotopic labeling of carbon dioxide in the off-gas was also measured by an on-line mass spectrometer. The (13)C-labeling data was then rigorously integrated for flux elucidation using the COMPLETE-MFA approach. The results provided important new insights into the metabolism of Geobacillus LC300, its efficient xylose utilization pathways, and the balance between carbon, redox and energy fluxes. The pentose phosphate pathway, glycolysis and TCA cycle were found to be highly active in Geobacillus LC300. The oxidative pentose phosphate pathway was also active and contributed significantly to NADPH production. No transhydrogenase activity was detected. Results from this work provide a solid foundation for future studies of this strain and its metabolic engineering and biotechnological applications.

  10. 13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry.

    PubMed

    Yuan, Yongbo; Yang, Tae Hoon; Heinzle, Elmar

    2010-07-01

    (13)C-based metabolic flux analysis ((13)CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured (13)C enrichment in proteinogenic amino acid hydrolyzates using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) from a series of parallel batch cultivations of Corynebacterium glutamicum utilizing mixtures of natural glucose and [1-(13)C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-(13)C] glucose. Decreasing the [1-(13)C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected (13)C enrichments in vivo fluxes in the central metabolism were determined by numerical optimization. The obtained flux distribution was very similar to those obtained from parallel labeling experiments using conventional high labeling GC-MS method and to published results. The GC-C-IRMS-based method involving low labeling degree of expensive tracer substrate, e.g. 1%, is well suited for larger laboratory and industrial pilot scale fermentations.

  11. Synthesis of [.sup.13C] and [.sup.2H] substituted methacrylic acid, [.sup.13C] and [.sup.2H] substituted methyl methacrylate and/or related compounds

    DOEpatents

    Alvarez, Marc A.; Martinez, Rodolfo A.; Unkefer, Clifford J.

    2008-01-22

    The present invention is directed to labeled compounds of the formulae ##STR00001## wherein Q is selected from the group consisting of --S--, --S(.dbd.O)--, and --S(.dbd.O).sub.2--, Z is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group selected from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each independently selected from the group consisting of a C.sub.1-C.sub.4 lower alkyl, an aryl, and an alkoxy group, and X is selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl group, and a fully-deuterated C.sub.1-C.sub.4 lower alkyl group. The present invention is also directed to a process of preparing labeled compounds, e.g., process of preparing [.sup.13C]methacrylic acid by reacting a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13CH.sub.2)-- aryl sulfone precursor with .sup.13CHI to form a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate, and, reacting the (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate with sodium hydroxide, followed by acid to form [.sup.13C]methacrylic acid. The present invention is further directed to a process of preparing [.sup.2H.sub.8]methyl methacrylate by reacting a (HOOC--C(C.sup.2H.sub.3).sub.2-- aryl sulfinyl intermediate with CD.sub.3I to form a (.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate, and heating the(.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate at temperatures and for time sufficient to form [.sup.2H.sub.8]methyl methacrylate.

  12. Extreme (13)C depletion of carbonates formed during oxidation of biogenic methane in fractured granite.

    PubMed

    Drake, Henrik; Åström, Mats E; Heim, Christine; Broman, Curt; Åström, Jan; Whitehouse, Martin; Ivarsson, Magnus; Siljeström, Sandra; Sjövall, Peter

    2015-01-01

    Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C13C as light as -69‰ V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13C-depleted carbonates ever reported, δ13C down to -125‰ V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane. PMID:25948095

  13. Extreme (13)C depletion of carbonates formed during oxidation of biogenic methane in fractured granite.

    PubMed

    Drake, Henrik; Åström, Mats E; Heim, Christine; Broman, Curt; Åström, Jan; Whitehouse, Martin; Ivarsson, Magnus; Siljeström, Sandra; Sjövall, Peter

    2015-05-07

    Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C13C as light as -69‰ V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13C-depleted carbonates ever reported, δ13C down to -125‰ V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane.

  14. Extreme 13C depletion of carbonates formed during oxidation of biogenic methane in fractured granite

    PubMed Central

    Drake, Henrik; Åström, Mats E.; Heim, Christine; Broman, Curt; Åström, Jan; Whitehouse, Martin; Ivarsson, Magnus; Siljeström, Sandra; Sjövall, Peter

    2015-01-01

    Precipitation of exceptionally 13C-depleted authigenic carbonate is a result of, and thus a tracer for, sulphate-dependent anaerobic methane oxidation, particularly in marine sediments. Although these carbonates typically are less depleted in 13C than in the source methane, because of incorporation of C also from other sources, they are far more depleted in 13C13C as light as −69‰ V-PDB) than in carbonates formed where no methane is involved. Here we show that oxidation of biogenic methane in carbon-poor deep groundwater in fractured granitoid rocks has resulted in fracture-wall precipitation of the most extremely 13C-depleted carbonates ever reported, δ13C down to −125‰ V-PDB. A microbial consortium of sulphate reducers and methane oxidizers has been involved, as revealed by biomarker signatures in the carbonates and S-isotope compositions of co-genetic sulphide. Methane formed at shallow depths has been oxidized at several hundred metres depth at the transition to a deep-seated sulphate-rich saline water. This process is so far an unrecognized terrestrial sink of methane. PMID:25948095

  15. Quantitative Analysis of Metabolic Mixtures by 2D 13C-Constant-Time TOCSY NMR Spectroscopy

    PubMed Central

    Bingol, Kerem; Zhang, Fengli; Bruschweiler-Li, Lei; Brüschweiler, Rafael

    2013-01-01

    An increasing number of organisms can be fully 13C-labeled, which has the advantage that their metabolomes can be studied by high-resolution 2D NMR 13C–13C constant-time (CT) TOCSY experiments. Individual metabolites can be identified via database searching or, in the case of novel compounds, through the reconstruction of their backbone-carbon topology. Determination of quantitative metabolite concentrations is another key task. Because significant peak overlaps in 1D NMR spectra prevents straightforward quantification through 1D peak integrals, we demonstrate here the direct use of 13C–13C CT-TOCSY spectra for metabolite quantification. This is accomplished through the quantum-mechanical treatment of the TOCSY magnetization transfer at short and long mixing times or by the use of analytical approximations, which are solely based on the knowledge of the carbon-backbone topologies. The methods are demonstrated for carbohydrate and amino-acid mixtures. PMID:23773204

  16. Conditions for (13)C NMR detection of 2-hydroxyglutarate in tissue extracts from isocitrate dehydrogenase-mutated gliomas.

    PubMed

    Pichumani, Kumar; Mashimo, Tomoyuki; Baek, Hyeon-Man; Ratnakar, James; Mickey, Bruce; DeBerardinis, Ralph J; Maher, Elizabeth A; Bachoo, Robert M; Malloy, Craig R; Kovacs, Zoltan

    2015-07-15

    (13)C NMR (nuclear magnetic resonance) spectroscopy of extracts from patient tumor samples provides rich information about metabolism. However, in isocitrate dehydrogenase (IDH)-mutant gliomas, (13)C labeling is obscured in oncometabolite 2-hydroxyglutaric acid (2 HG) by glutamate and glutamine, prompting development of a simple method to resolve the metabolites. J-coupled multiplets in 2 HG were similar to glutamate and glutamine and could be clearly resolved at pH 6. A cryogenically cooled (13)C probe, but not J-resolved heteronuclear single quantum coherence spectroscopy, significantly improved detection of 2 HG. These methods enable the monitoring of (13)C-(13)C spin-spin couplings in 2 HG expressing IDH-mutant gliomas.

  17. 13C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H

    PubMed Central

    Ostermann, Steffen; Richhardt, Janine; Bringer, Stephanie; Bott, Michael; Wiechert, Wolfgang; Oldiges, Marco

    2015-01-01

    Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner–Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically 13C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from 13C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with 13C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments. PMID:26404385

  18. NMR analyses of the cold cataract. III. /sup 13/C acrylamide studies

    SciTech Connect

    Lerman, S.; Megaw, J.M.; Moran, M.N.

    1985-10-01

    /sup 13/C-enriched acrylamide was employed to further delineate the action of this compound in preventing the cold cataract phenomenon when it is incorporated (in vitro) into young human and rabbit lenses. The extent of acrylamide incorporation, in the dark and with concurrent UV exposure, was monitored by /sup 13/C NMR spectroscopy. These studies provide further evidence that UV exposure causes permanent acrylamide photobinding within the lens. In such lenses, the gamma crystallin fraction of the soluble lens proteins is affected to the greatest extent. It appears to become aggregated and/or combined with the alpha and beta fractions resulting in an apparent loss of most of the gamma monomers. There is also an age-related effect with respect to the amount of acrylamide that can be incorporated into the lens. The decrease in acrylamide incorporation with age directly parallels the age-related decline in gamma crystallin levels.

  19. Novel Imaging Contrast Methods for Hyperpolarized 13 C Magnetic Resonance Imaging

    NASA Astrophysics Data System (ADS)

    Reed, Galen Durant

    Magnetic resonance imaging using hyperpolarized 13C-labeled small molecules has emerged as an extremely powerful tool for the in vivo monitoring of perfusion and metabolism. This work presents methods for improved imaging, parameter mapping, and image contrast generation for in vivo hyperpolarized 13C MRI. Angiography using hyperpolarized urea was greatly improved with a highly T2-weighted acquisition in combination with 15N labeling of the urea amide groups. This is due to the fact that the T2 of [13C]urea is strongly limited by the scalar coupling to the neighboring quadrupolar 14N. The long in vivo T2 values of [13C, 15N2]urea were utilized for sub-millimeter projection angiography using a contrast agent that could be safely injected in concentrations of 10-100 mM while still tolerated in patients with renal insufficiency. This study also presented the first method for in vivo T2 mapping of hyperpolarized 13C compounds. The in vivo T2 of urea was short in the blood and long within the kidneys. This persistent signal component was isolated to the renal filtrate, thus enabling for the first time direct detection of an imaging contrast agent undergoing glomerular filtration. While highly T2-weighted acquisitions select for molecules with short rotational correlation times, high diffusion weighting selects for those with the long translational correlation times. A specialized spin-echo EPI sequence was developed in order to generate highly diffusion-weighted hyperpolarized 13C images on a clinical MRI system operating within clinical peak- RF and gradient amplitude constraints. Low power adiabatic spin echo pulses were developed in order to generate a sufficiently large refocused bandwidth while maintaining low nominal power. This diffusion weighted acquisition gave enhanced tumor contrast-to-noise ratio when imaging [1-13C]lactate after infusion of [1-13C]pyruvate. Finally, the first in-man hyperpolarized 13C MRI clinical trial is discussed.

  20. In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.

    PubMed

    Kanje, Sara; Hober, Sophia

    2015-04-01

    Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.

  1. Double cross polarization /sup 13/C-NMR experiment in solid fossil fuel structure analysis

    SciTech Connect

    Hagaman, E.W.; Woody, M.C.

    1988-01-01

    The Double Cross Polarization /sup 13/C-MAS/NMR experiment has been used to derive a new operational classification of solid fossil fuels based on chemical reactivity. The method requires labeling reactive sites in the organic matrix with a magnetically active isotope not present in the precursor material, and using the local, isolated dipole-dipole interaction between this nucleus and nearby /sup 13/C nuclei to detect via cross polarization the carbon centers in the vicinity of the label. The technique is a marriage of chemistry and spectroscopy and the information content of the DCP spectra is defined by both partners. /sup 1/H-/sup 13/C-/sup 31/P DCP/MAS /sup 13/C-NMR spectroscopy has been used to statistically describe phenolic ortho-substitution patterns of coals via their aryl phosphinate or phosphate derivatives. In these applications of DCP NMR the new, detailed structure and/or reactivity information is realized by detection of carbon resonances one or more bonds removed from the reaction center, but in a volume element of intramolecular dimensions. To the extent that intermolecular contributions to the spectrum are detected, and not recognized as such, the structure/reactivity correlation is weakened. Direct substitution of phosphorus on the aromatic rings in the organic matrix of the coal is not readily accomplished. This environment potentially can be labeled with fluorine in a selective fashion using newly developed reagents. The possibility of determining the changes in average ring substitution patterns as a function of chemical treatment or coal diagenesis emerges. Recent developments in the field of DCP /sup 13/C NMR are presented.

  2. Dynamic nuclear polarization-enhanced 1H–13C double resonance NMR in static samples below 20 K

    PubMed Central

    Potapov, Alexey; Thurber, Kent R.; Yau, Wai-Ming; Tycko, Robert

    2012-01-01

    We demonstrate the feasibility of one-dimensional and two-dimensional 1H–13C double resonance NMR experiments with dynamic nuclear polarization (DNP) at 9.4 T and temperatures below 20 K, including both 1H–13C cross-polarization and 1H decoupling, and discuss the effects of polarizing agent type, polarizing agent concentration, temperature, and solvent deuteration. We describe a two-channel low-temperature DNP/NMR probe, capable of carrying the radio-frequency power load required for 1H–13C cross-polarization and high-power proton decoupling. Experiments at 8 K and 16 K reveal a significant T2 relaxation of 13C, induced by electron spin flips. Carr–Purcell experiments and numerical simulations of Carr–Purcell dephasing curves allow us to determine the effective correlation time of electron flips under our experimental conditions. The dependence of the DNP signal enhancement on electron spin concentration shows a maximum near 80 mM. Although no significant difference in the absolute DNP enhancements for triradical (DOTOPA-TEMPO) and biradical (TOTAPOL) dopants was found, the triradical produced greater DNP build-up rates, which are advantageous for DNP experiments. Additionally the feasibility of structural measurements on 13C-labeled biomolecules was demonstrated with a two-dimensional 13C–13C exchange spectrum of selectively 13C-labeled β-amyloid fibrils. PMID:22743540

  3. Critical appraisal of 13C breath tests for microsomal liver function: aminopyrine revisited.

    PubMed

    Pijls, Kirsten E; de Vries, Hanne; Nikkessen, Suzan; Bast, Aalt; Wodzig, Will K W H; Koek, Ger H

    2014-04-01

    As liver diseases are a major health problem and especially the incidence of metabolic liver diseases like non-alcoholic fatty liver disease (NAFLD) is rising, the demand for non-invasive tests is growing to replace liver biopsy. Non-invasive tests such as carbon-labelled breath tests can provide a valuable contribution to the evaluation of metabolic liver function. This review aims to critically appraise the value of the (13) C-labelled microsomal breath tests for the evaluation of metabolic liver function, and to discuss the role of cytochrome P450 enzymes in the metabolism of the different probe drugs, especially of aminopyrine. Although a number of different probe drugs have been used in breath tests, the perfect drug to assess the functional metabolic capacity of the liver has not been found. Data suggest that both the (13) C(2) -aminopyrine and the (13) C-methacetin breath test can play a role in assessing the capacity of the microsomal liver function and may be useful in the follow-up of patients with chronic liver diseases. Furthermore, CYP2C19 seems to be an important enzyme in the N-demethylation of aminopyrine, and polymorphisms in this gene may influence breath test values, which should be kept in mind when performing the (13) C(2) -aminopyrine breath test in clinical practice.

  4. Methylation patterns of aquatic humic substances determined by 13C NMR spectroscopy

    USGS Publications Warehouse

    Thorn, K.A.; Steelink, C.; Wershaw, R. L.

    1987-01-01

    13C NMR spectroscopy is used to examine the hydroxyl group functionality of a series of humic and fulvic acids from different aquatic environments. Samples first are methylated with 13C-labeled diazomethane. The NMR spectra of the diazomethylated samples allow one to distinguish between methyl esters of carboxylic acids, methyl ethers of phenolic hydroxyls, and methyl ethers of phenolic hydroxyls adjacent to two substituents. Samples are then permethylated with 13C-labeled methyl iodide/NaH. 13C NMR spectra of permethylated samples show that a significant fraction of the hydroxyl groups is not methylated with diazomethane alone. In these spectra methyl ethers of carbohydrate and aliphatic hydroxyls overlap with methyl ethers of phenolic hydroxyls. Side reactions of the methyltion procedure including carbon methylation in the CH3I/NaH procedure, are also examined. Humic and fulvic acids from bog, swamp, groundwater, and lake waters showssome differences in their distribution of hydroxyl groups, mainly in the concentrations of phenolic hydroxyls, which may be attributed to their different biogeochemical origins. ?? 1987.

  5. (Bio)degradation of glyphosate in water-sediment microcosms - A stable isotope co-labeling approach.

    PubMed

    Wang, Shizong; Seiwert, Bettina; Kästner, Matthias; Miltner, Anja; Schäffer, Andreas; Reemtsma, Thorsten; Yang, Qi; Nowak, Karolina M

    2016-08-01

    Glyphosate and its metabolite aminomethylphosphonic acid (AMPA) are frequently detected in water and sediments. Up to date, there are no comprehensive studies on the fate of glyphosate in water-sediment microcosms according to OECD 308 guideline. Stable isotope co-labeled (13)C3(15)N-glyphosate was used to determine the turnover mass balance, formation of metabolites, and formation of residues over a period of 80 days. In the water-sediment system, 56% of the initial (13)C3-glyphosate equivalents was ultimately mineralized, whereas the mineralization in the water system (without sediment) was low, reaching only 2% of (13)C-glyphosate equivalents. This finding demonstrates the key role of sediments in its degradation. Glyphosate was detected below detection limit in the water compartment on day 40, but could still be detected in the sediments, ultimately reaching 5% of (13)C3(15)N-glyphosate equivalents. A rapid increase in (13)C(15)N-AMPA was noted after 10 days, and these transformation products ultimately constituted 26% of the (13)C3-glyphosate equivalents and 79% of the (15)N-glyphosate equivalents. In total, 10% of the (13)C label and 12% of the (15)N label were incorporated into amino acids, indicating no risk bearing biogenic residue formation from (13)C3(15)N-glyphosate. Initially, glyphosate was biodegraded via the sarcosine pathway related to microbial growth, as shown by co-labeled (13)C(15)N-glycine and biogenic residue formation. Later, degradation via AMPA dominated under starvation conditions, as shown by the contents of (13)C-glycine. The presented data provide the first evidence of the speciation of the non-extractable residues as well as the utilization of glyphosate as a carbon and nitrogen source in the water-sediment system. This study also highlights the contribution of both the sarcosine and the AMPA degradation pathways under these conditions. PMID:27140906

  6. (Bio)degradation of glyphosate in water-sediment microcosms - A stable isotope co-labeling approach.

    PubMed

    Wang, Shizong; Seiwert, Bettina; Kästner, Matthias; Miltner, Anja; Schäffer, Andreas; Reemtsma, Thorsten; Yang, Qi; Nowak, Karolina M

    2016-08-01

    Glyphosate and its metabolite aminomethylphosphonic acid (AMPA) are frequently detected in water and sediments. Up to date, there are no comprehensive studies on the fate of glyphosate in water-sediment microcosms according to OECD 308 guideline. Stable isotope co-labeled (13)C3(15)N-glyphosate was used to determine the turnover mass balance, formation of metabolites, and formation of residues over a period of 80 days. In the water-sediment system, 56% of the initial (13)C3-glyphosate equivalents was ultimately mineralized, whereas the mineralization in the water system (without sediment) was low, reaching only 2% of (13)C-glyphosate equivalents. This finding demonstrates the key role of sediments in its degradation. Glyphosate was detected below detection limit in the water compartment on day 40, but could still be detected in the sediments, ultimately reaching 5% of (13)C3(15)N-glyphosate equivalents. A rapid increase in (13)C(15)N-AMPA was noted after 10 days, and these transformation products ultimately constituted 26% of the (13)C3-glyphosate equivalents and 79% of the (15)N-glyphosate equivalents. In total, 10% of the (13)C label and 12% of the (15)N label were incorporated into amino acids, indicating no risk bearing biogenic residue formation from (13)C3(15)N-glyphosate. Initially, glyphosate was biodegraded via the sarcosine pathway related to microbial growth, as shown by co-labeled (13)C(15)N-glycine and biogenic residue formation. Later, degradation via AMPA dominated under starvation conditions, as shown by the contents of (13)C-glycine. The presented data provide the first evidence of the speciation of the non-extractable residues as well as the utilization of glyphosate as a carbon and nitrogen source in the water-sediment system. This study also highlights the contribution of both the sarcosine and the AMPA degradation pathways under these conditions.

  7. Measuring long-lived 13C2 state lifetimes at natural abundance

    NASA Astrophysics Data System (ADS)

    Claytor, Kevin; Theis, Thomas; Feng, Yesu; Warren, Warren

    2014-02-01

    Long-lived disconnected eigenstates (for example, the singlet state in a system with two nearly equivalent carbons, or the singlet-singlet state in a system with two chemically equivalent carbons and two chemically equivalent hydrogens) hold the potential to drastically extend the lifetime of hyperpolarization in molecular tracers for in vivo magnetic resonance imaging (MRI). However, a first-principles calculation of the expected lifetime (and thus selection of potential imaging agents) is made very difficult because of the large variety of relevant intra- and intermolecular relaxation mechanisms. As a result, all previous measurements relied on costly and time consuming syntheses of 13C labeled compounds. Here we show that it is possible to determine 13C singlet state lifetimes by detecting the naturally abundant doubly-labeled species. This approach allows for rapid and low cost screening of potential molecular biomarkers bearing long-lived states.

  8. Identification of metabolically active bacteria in the gut of the generalist Spodoptera littoralis via DNA stable isotope probing using 13C-glucose.

    PubMed

    Shao, Yongqi; Arias-Cordero, Erika M; Boland, Wilhelm

    2013-01-01

    Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction(1), boosting the immune response(2), pheromone production(3), as well as nutrition, including the synthesis of essential amino acids(4,) among others.     Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms. To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing (13)C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA(5). The incorporation of (13)C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled ((12)C) one. In the end, the (13)C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the (12)C-unlabeled similar one(6). Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species. Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The

  9. Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

    PubMed Central

    Boland, Wilhelm

    2013-01-01

    Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others.     Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms. To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species. Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA

  10. Functional groups identified by solid state 13C NMR spectroscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Animal manure is generally high in organic matter intensity so it is well suitable for 13C nuclear magnetic resonance (NMR) analysis. Solid-state 13C NMR techniques used in characterizing organic matter and its components include, but are not limited to, cross-polarization /magic angle spinning (CP...

  11. The use of isotope ratios (13C/12C) for vegetable oils authentication

    NASA Astrophysics Data System (ADS)

    Cristea, G.; Magdas, D. A.; Mirel, V.

    2012-02-01

    Stable isotopes are now increasingly used for the control of the geographical origin or authenticity of food products. The falsification may be more or less sophisticated and its sophistication as well as its costs increases with the improvement of analytical methods. In this study 22 vegetable oils (olive, sunflower, palm, maize) commercialized on Romanian market were investigated by mean of δ13C in bulk oil and the obtained results were compared with those reported in literature in order to check the labeling of these natural products. The obtained results were in the range of the mean values found in the literature for these types of oils, thus providing their accurate labeling.

  12. Partial 13C isotopic enrichment of nucleoside monophosphates: useful reporters for NMR structural studies

    PubMed Central

    Kishore, Anita I.; Mayer, Michael R.; Prestegard, James H.

    2005-01-01

    Analysis of the 13C isotopic labeling patterns of nucleoside monophosphates (NMPs) extracted from Escherichia coli grown in a mixture of C-1 and C-2 glucose is presented. By comparing our results to previous observations on amino acids grown in similar media, we have been able to rationalize the labeling pattern based on the well-known biochemistry of nucleotide biosynthesis. Except for a few notable absences of label (C4 in purines and C3′ in ribose) and one highly enriched site (C1′ in ribose), most carbons are randomly enriched at a low level (an average of 13%). These sparsely labeled NMPs give less complex NMR spectra than their fully isotopically labeled analogs due to the elimination of most 13C–13C scalar couplings. The spectral simplicity is particularly advantageous when working in ordered systems, as illustrated with guanosine diphosphate (GDP) bound to ADP ribosylation factor 1 (ARF1) aligned in a liquid crystalline medium. In this system, the absence of scalar couplings and additional long-range dipolar couplings significantly enhances signal to noise and resolution. PMID:16254075

  13. Analysis of mutational lesions of acetate metabolism in Neurospora crassa by 13C nuclear magnetic resonance.

    PubMed Central

    Thomas, G H; Baxter, R L

    1987-01-01

    The adaptation of Neurospora crassa mycelium to growth on acetate as the sole carbon source was examined by using 13C nuclear magnetic resonance. Extracts were examined by nuclear magnetic resonance at various times after transfer of the mycelium from medium containing sucrose to medium containing [2-13C]acetate as the sole carbon source. The label was initially seen to enter the alanine, glutamate, and glutamine pools, and after 6 h 13C-enriched trehalose was evident, indicating that gluconeogenesis was occurring. Analysis of the isotopomer ratios in the alanine and glutamate-glutamine pools indicated that substantial glyoxylate cycle activity became evident between 2 and 4 h after transfer. Immediately after transfer of the mycelium to acetate medium, the alanine pool increased to about four times its previous level, only a small fraction of which was enriched with 13C. The quantity of 13C-enriched alanine remained almost constant between 2 and 7.5 h after the transfer, whereas the overall alanine pool decreased to its original level. The selective catabolism of the unenriched alanine leads us to suggest that the alanine pool is partitioned into two compartments during adaptation. Two acetate-nonutilizing mutants were also studied by this technique. An acu-3 strain, deficient for isocitrate lyase (EC 4.1.3.1) activity, showed metabolic changes consistent with this lesion. An acp strain, previously thought to be deficient in an inducible acetate permease, took up [2-13C]acetate but showed no evidence of glyoxylate cycle activity despite synthesizing the necessary enzymes; the lesion was therefore reinterpreted. PMID:2947898

  14. Whole-core analysis by sup 13 C NMR

    SciTech Connect

    Vinegar, H.J.; Tutunjian, P.N. ); Edelstein, W.A.; Roemer, P.B. )

    1991-06-01

    This paper reports on a whole-core nuclear magnetic resonance (NMR) system that was used to obtain natural abundance {sup 13}C spectra. The system enables rapid, nondestructive measurements of bulk volume of movable oil, aliphatic/aromatic ratio, oil viscosity, and organic vs. carbonate carbon. {sup 13}C NMR can be used in cores where the {sup 1}H NMR spectrum is too broad to resolve oil and water resonances separately. A 5 1/4-in. {sup 13}C/{sup 1}H NMR coil was installed on a General Electric (GE) CSI-2T NMR imager/spectrometer. With a 4-in.-OD whole core, good {sup 13}C signal/noise ratio (SNR) is obtained within minutes, while {sup 1}H spectra are obtained in seconds. NMR measurements have been made of the {sup 13}C and {sup 1}H density of crude oils with a wide range of API gravities. For light- and medium-gravity oils, the {sup 13}C and {sup 1}H signal per unit volume is constant within about 3.5%. For heavy crudes, the {sup 13}C and {sup 1}H density measured by NMR is reduced by the shortening of spin-spin relaxation time. {sup 13}C and {sup 1}H NMR spin-lattice relaxation times were measured on a suite of Cannon viscosity standards, crude oils (4 to 60{degrees} API), and alkanes (C{sub 5} through C{sub 16}) with viscosities at 77{degrees}F ranging from 0.5 cp to 2.5 {times} 10{sup 7} cp. The {sup 13}C and {sup 1}H relaxation times show a similar correlation with viscosity from which oil viscosity can be estimated accurately for viscosities up to 100 cp. The {sup 13}C surface relaxation rate for oils on water-wet rocks is very low. Nonproton decoupled {sup 13}C NMR is shown to be insensitive to kerogen; thus, {sup 13}C NMR measures only the movable hydrocarbon content of the cores. In carbonates, the {sup 13}C spectrum also contains a carbonate powder pattern useful in quantifying inorganic carbon and distinguishing organic from carbonate carbon.

  15. Detection of intracellular lactate with localized diffusion { 1H- 13C}-spectroscopy in rat glioma in vivo

    NASA Astrophysics Data System (ADS)

    Pfeuffer, Josef; Lin, Joseph C.; DelaBarre, Lance; Ugurbil, Kamil; Garwood, Michael

    2005-11-01

    The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of 13C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4 T (400 MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM ('adiabatic carbon editing and decoupling'). 13C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, { 1H- 13C}-lactate and { 1H- 13C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into { 1H- 13C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50 ms/μm 2). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, 1H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([ 13C] lactate) originates from an intracellular compartment.

  16. Identification of biomass utilizing bacteria in a carbon-depleted glacier forefield soil by the use of 13C DNA stable isotope probing.

    PubMed

    Zumsteg, Anita; Schmutz, Stefan; Frey, Beat

    2013-06-01

    As Alpine glaciers are retreating rapidly, bare soils with low organic C and N contents are becoming exposed. Carbon availability is a key factor regulating microbial diversity and ecosystem functioning in these soils. The aim of this study was to investigate how bacterial activity, community structure and composition are influenced by organic carbon availability. Bare soils were supplied with (13)C-labelled fungal (Penicillium sp.) and green algal (Chlorella sp.) biomass and the CO2 evolution and its δ(13)C signature were monitored up to 60 days. These organisms have previously been isolated near the glacier terminus. DNA stable isotope probing followed by T-RFLP profiling and sequencing of 16S rRNA genes was employed to identify consumers able to assimilate carbon from these biomass amendments. Higher respiration and higher bacterial activity indicated a more efficient utilization of algal cells than fungal cells. Flavobacterium sp. predominantly incorporated fungal-derived C, whereas the algal-derived C was mainly incorporated by Acidobacteria and Proteobacteria. This study emphasizes the important role of both fungal and algal biomass in increasing the carbon pool in recently deglaciated bare soils, as only 20% of the added C was respired as CO2, and the rest, we presume, remained in the soil.

  17. Chain-Folding Structures of a Semi-crystalline Polymer in Bulk and Single Crystals Elucidated by 13C-13C Double Quantum NMR

    NASA Astrophysics Data System (ADS)

    Hong, You-Lee; Miyoshi, Toshikazu

    2014-03-01

    Semi-crystalline polymers are crystallized as folded chains in thin lamellae of ca. 5-20 nm from random coils in the melt and solution states. However, understanding of detailed chain-folding structure and crystallization mechanism are still challenging issue due to various experimental limitations. We recently developed a new strategy using 13C-13C double-quantum (DQ) NMR with selectively 13C isotope labeled isotactic poly(1-butene) form I to investigate chain-trajectory in solution and melt grown crystals at various Tcs. This new method can determine the re-entrance sites, the successive folding number (n) , and the fractions (F) of chain-folding in a wide Tc range. In melt grown crystals at Tc = 95 °C, a comparison of experimental and simulated DQ efficiency determined that the polymer chains alternatively change chain-folding directions and the stems tightly pack via intramolecular interactions, and the fraction (F) of adjacent re-entry structure ranges from 70% at n = 4 to 100% at mixed structures of n = 1 and 2. Furthermore, DQ efficiency is independent of Tc in bulk crystals. This means chain-folding do not change in a wide Tcs. DMR-1105829.

  18. 13C metabolic flux analysis at a genome-scale.

    PubMed

    Gopalakrishnan, Saratram; Maranas, Costas D

    2015-11-01

    Metabolic models used in 13C metabolic flux analysis generally include a limited number of reactions primarily from central metabolism. They typically omit degradation pathways, complete cofactor balances, and atom transition contributions for reactions outside central metabolism. This study addresses the impact on prediction fidelity of scaling-up mapping models to a genome-scale. The core mapping model employed in this study accounts for (75 reactions and 65 metabolites) primarily from central metabolism. The genome-scale metabolic mapping model (GSMM) (697 reaction and 595 metabolites) is constructed using as a basis the iAF1260 model upon eliminating reactions guaranteed not to carry flux based on growth and fermentation data for a minimal glucose growth medium. Labeling data for 17 amino acid fragments obtained from cells fed with glucose labeled at the second carbon was used to obtain fluxes and ranges. Metabolic fluxes and confidence intervals are estimated, for both core and genome-scale mapping models, by minimizing the sum of square of differences between predicted and experimentally measured labeling patterns using the EMU decomposition algorithm. Overall, we find that both topology and estimated values of the metabolic fluxes remain largely consistent between core and GSM model. Stepping up to a genome-scale mapping model leads to wider flux inference ranges for 20 key reactions present in the core model. The glycolysis flux range doubles due to the possibility of active gluconeogenesis, the TCA flux range expanded by 80% due to the availability of a bypass through arginine consistent with labeling data, and the transhydrogenase reaction flux was essentially unresolved due to the presence of as many as five routes for the inter-conversion of NADPH to NADH afforded by the genome-scale model. By globally accounting for ATP demands in the GSMM model the unused ATP decreased drastically with the lower bound matching the maintenance ATP requirement. A non

  19. Study of Urban environmental quality through Isotopes δ13C

    NASA Astrophysics Data System (ADS)

    González-Sosa, E.; Mastachi-Loza, C.; Becerril-Piña, R.; Ramos-Salinas, N. M.

    2012-04-01

    Usually, trees with similar pH values on their bark develop epiphytes of similar species, the acidity to be a factor for growth. The aim of the study was evaluate the air quality through isotope δ13C in order to define the levels of environmental quality in the city of Queretaro, Mexico. In this work were collected at least 4 epiphytes positioned in trees of the species Prosopis Laevigata at 25 sites of Queretaro City. The samples were analyzed for trace elements with an inductively coupled plasma atomic emission spectroscopy (ICP). The collecting took place during dry period, in May and early rain June 2011 period, and on four sectors to identify the spatial distribution of pollution, using isotopic analysis of concentration of δ 13C. According with the results there are significant differences among the species in each of the sampled areas. The 5 February Avenue presented greater diversity and richness of δ13C, followed by those who were surveyed in the proximity of the UAQ and finally in the middle-east area. An average value of δ13C-17.92%, followed by those surveyed in the vicinity of the UAQ that correspond to sector I and II with an concentration of δ13C-17.55% and δ13C-17.22%, and finally the samples collected in trees scattered in the East-Sector II and IV with a value of δ13C-17.02% and δ13C-15.62%, respectively. Also were observed differences between the dry and wet period. It is likely that these results of δ 13C in moist period reflect the drag of the isotopes due to rain events that could mark a trend in the dilution of this element, however there is a trend in terms of abundance and composition of finding more impact in those species sampled in dry period, in May and early June 2011.

  20. {sup 13}C chemical shift anisotropies for carbonate ions in cement minerals and the use of {sup 13}C, {sup 27}Al and {sup 29}Si MAS NMR in studies of Portland cement including limestone additions

    SciTech Connect

    Sevelsted, Tine F.; Herfort, Duncan

    2013-10-15

    {sup 13}C isotropic chemical shifts and chemical shift anisotropy parameters have been determined for a number of inorganic carbonates relevant in cement chemistry from slow-speed {sup 13}C MAS or {sup 13}C({sup 1}H) CP/MAS NMR spectra (9.4 T or 14.1 T) for {sup 13}C in natural abundance. The variation in the {sup 13}C chemical shift parameters is relatively small, raising some doubts that different carbonate species in Portland cement-based materials may not be sufficiently resolved in {sup 13}C MAS NMR spectra. However, it is shown that by combining {sup 13}C MAS and {sup 13}C({sup 1}H) CP/MAS NMR carbonate anions in anhydrous and hydrated phases can be distinguished, thereby providing valuable information about the reactivity of limestone in cement blends. This is illustrated for three cement pastes prepared from an ordinary Portland cement, including 0, 16, and 25 wt.% limestone, and following the hydration for up to one year. For these blends {sup 29}Si MAS NMR reveals that the limestone filler accelerates the hydration for alite and also results in a smaller fraction of tetrahedrally coordinated Al incorporated in the C-S-H phase. The latter result is more clearly observed in {sup 27}Al MAS NMR spectra of the cement–limestone blends and suggests that dissolved aluminate species in the cement–limestone blends readily react with carbonate ions from the limestone filler, forming calcium monocarboaluminate hydrate. -- Highlights: •{sup 13}C chemical shift anisotropies for inorganic carbonates from {sup 13}C MAS NMR. •Narrow {sup 13}C NMR chemical shift range (163–171 ppm) for inorganic carbonates. •Anhydrous and hydrated carbonate species by {sup 13}C MAS and {sup 13}C({sup 1}H) CP/MAS NMR. •Limestone accelerates the hydration for alite in Portland – limestone cements. •Limestone reduces the amount of aluminium incorporated in the C-S-H phase.

  1. Relative contributions of rhizosphere and microbial respiration to belowground and total ecosystem respiration in arctic tussock tundra: results of a 13C pulse-chase experiment

    NASA Astrophysics Data System (ADS)

    Segal, A. D.; Sullivan, P.; Weintraub, M. N.; Darrouzet-Nardi, A.; Steltzer, H.

    2011-12-01

    Many arctic ecosystems that have historically been strong carbon (C) sinks are becoming sources of C to the atmosphere. Although ecosystem respiration is the largest C flux out of ecosystems, our ability to model respiration lags considerably behind our ability to model photosynthesis in the Arctic. Understanding the controls on respiration is especially important for an ecosystem which appears to be experiencing the greatest climate warming and also contains large stores of soil C. Partitioning respiration into its component fluxes and identifying factors controlling respiration of each component is a critical first step towards improving our ability to model changes in respiration. However, partitioning belowground constituents has proven to be challenging in most ecosystems. Therefore, to accurately estimate rhizosphere respiration and bulk soil microbial respiration in moist acidic tussock tundra, we selected an isotopic method that results in minimal disturbance of belowground processes. In mid July of 2011, we introduced a 13CO2 label into a clear ecosystem CO2 flux chamber, allowed the vegetation to incorporate the label through photosynthesis and returned 2 days and 4 days after labeling to follow the movement of the 13C signal. A smaller CO2 flux chamber was used to chase the label separately in tussock and inter-tussock areas. All above ground plant tissue was clipped immediately before the chase measurements and soil cores were taken immediately after chasing the label. Syringe samples (n=5 or 6) were collected from the small flux chamber at regular intervals as CO2 concentrations were allowed to build, and Keeling plots were used to estimate δ13C of belowground respiration. After completing the field measurements, the soil cores were sorted into live roots and root free soil. Samples of each were incubated in mason jars placed in a 10°C water bath. The jars were scrubbed free of CO2 and syringe samples were collected from each jar after CO2

  2. /sup 1/H and /sup 13/C spin-lattice relaxation in gaseous benzene

    SciTech Connect

    Folkendt, M.M.; Weiss-Lopez, B.E.; True, N.S.

    1988-08-25

    The nuclear spin-lattice relaxation time, T/sub 1/, measured for benzene protons at densities between 0.81 and 54.4 mol/m/sup 3/ (15 and 980 Torr) at 381 K exhibits a characteristic nonlinear density dependence. Analysis of the density-dependent T/sub 1/ data yields a spin-rotation coupling constant, C/sub eff/, of /vert bar/182.6 (0.4)/vert bar/ Hz and an angular momentum reorientation cross section, sigma, of 131 (1) /Angstrom//sup 2/. The /sup 13/C spin-lattice relaxation time of singly labeled /sup 13/C benzene is a linear function of density over the density range 1.07-75.12 mol/m/sup 3/ (20-1330 Torr). /sup 13/C T/sub 1/ values are shorter than /sup 1/H T/sub 1/ values by a factor of ca. 100 at comparable densities. The nuclear Overhauser enhancement factor, /eta/, is 0.0 /plus minus/ 0.02 at densities between 11 and 85.3 mol/m/sup 3/ (200 and 1500 Torr), demonstrating that dipole-dipole relaxation is relatively inefficient in this region. The spin-rotation coupling constant, C/sub eff/, for /sup 13/C nuclei in benzene is estimated to be /vert bar/1602 (68)/vert bar/ Hz.

  3. 13CFLUX2—high-performance software suite for 13C-metabolic flux analysis

    PubMed Central

    Weitzel, Michael; Nöh, Katharina; Dalman, Tolga; Niedenführ, Sebastian; Stute, Birgit; Wiechert, Wolfgang

    2013-01-01

    Summary: 13C-based metabolic flux analysis (13C-MFA) is the state-of-the-art method to quantitatively determine in vivo metabolic reaction rates in microorganisms. 13CFLUX2 contains all tools for composing flexible computational 13C-MFA workflows to design and evaluate carbon labeling experiments. A specially developed XML language, FluxML, highly efficient data structures and simulation algorithms achieve a maximum of performance and effectiveness. Support of multicore CPUs, as well as compute clusters, enables scalable investigations. 13CFLUX2 outperforms existing tools in terms of universality, flexibility and built-in features. Therewith, 13CFLUX2 paves the way for next-generation high-resolution 13C-MFA applications on the large scale. Availability and implementation: 13CFLUX2 is implemented in C++ (ISO/IEC 14882 standard) with Java and Python add-ons to run under Linux/Unix. A demo version and binaries are available at www.13cflux.net. Contact: info@13cflux.net or k.noeh@fz-juelich.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23110970

  4. 13C-Breath Tests for Sucrose Digestion in Congenital Sucrase Isomaltase Deficient and Sacrosidase Supplemented Patients

    PubMed Central

    Robayo-Torres, Claudia C.; Opekun, Antone R.; Quezada-Calvillo, Roberto; Xavier, Villa; Smith, E. O’Brian; Navarrete, Marilyn; Baker, S. Susan; Nichols, Buford L

    2008-01-01

    Congenital sucrase-isomaltase deficiency (CSID) is characterized by absence or deficiency of the mucosal sucrase-isomaltase enzyme. Specific diagnosis requires upper gastrointestinal biopsy with evidence of low to absent sucrase enzyme activity and normal histology. The hydrogen breath test (BT) is useful but is not specific for confirmation of CSID. We investigated a more specific 13C-sucrose labeled BT. Objectives were to determine if CSID can be detected with the 13C-sucrose BT without duodenal biopsy sucrase assay and if the 13C-sucrose BT can document restoration of sucrose digestion by CSID patients after oral supplementation with sacrosidase (Sucraid®). Methods Ten CSID patients were diagnosed by low biopsy sucrase activity. Ten controls were children who underwent endoscopy and biopsy because of dyspepsia or chronic diarrhea with normal mucosal enzymes activity and histology. Uniformly-labeled 13C-glucose and 13C-sucrose loads were orally administered. 13CO2 breath enrichments were assayed using an infrared spectrophotometer. In CSID patients the 13C-sucrose load was repeated adding Sucraid®. Sucrose digestion and oxidation were calculated as a mean % coefficient of glucose oxidation (% CGO) averaged between 30 and 90 minutes. Results Classification of patients by 13C-sucrose BT % CGO agreed with biopsy sucrase activity. The breath test also documented the return to normal of sucrose digestion and oxidation after supplementation of CSID patients with Sucraid®. Conclusion 13C-sucrose BT is an accurate and specific non-invasive confirmatory test for CSID and for enzyme replacement management. PMID:19330928

  5. Characterization of cerebral glutamine uptake from blood in the mouse brain: implications for metabolic modeling of 13C NMR data

    PubMed Central

    Bagga, Puneet; Behar, Kevin L; Mason, Graeme F; De Feyter, Henk M; Rothman, Douglas L; Patel, Anant B

    2014-01-01

    13C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-13C]/[1,6-13C2]glucose as labeled substrate have consistently found a lower enrichment (∼25% to 30%) of glutamine-C4 compared with glutamate-C4 at isotopic steady state. The source of this isotope dilution has not been established experimentally but may potentially arise either from blood/brain exchange of glutamine or from metabolism of unlabeled substrates in astrocytes, where glutamine synthesis occurs. In this study, the contribution of the former was evaluated ex vivo using 1H-[13C]-NMR spectroscopy together with intravenous infusion of [U-13C5]glutamine for 3, 15, 30, and 60 minutes in mice. 13C labeling of brain glutamine was found to be saturated at plasma glutamine levels >1.0 mmol/L. Fitting a blood–astrocyte–neuron metabolic model to the 13C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, VGln(in), 0.036±0.002 μmol/g per minute for plasma glutamine of 1.8 mmol/L. For physiologic plasma glutamine level (∼0.6 mmol/L), VGln(in) would be ∼0.010 μmol/g per minute, which corresponds to ∼6% of the glutamine synthesis rate and rises to ∼11% for saturating blood glutamine concentrations. Thus, glutamine influx from blood contributes at most ∼20% to the dilution of astroglial glutamine-C4 consistently seen in metabolic studies using [1-13C]glucose. PMID:25074745

  6. Detection of inflammatory cell function using 13C magnetic resonance spectroscopy of hyperpolarized [6-13C]-arginine

    PubMed Central

    Najac, Chloé; Chaumeil, Myriam M.; Kohanbash, Gary; Guglielmetti, Caroline; Gordon, Jeremy W.; Okada, Hideho; Ronen, Sabrina M.

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are highly prevalent inflammatory cells that play a key role in tumor development and are considered therapeutic targets. MDSCs promote tumor growth by blocking T-cell-mediated anti-tumoral immune response through depletion of arginine that is essential for T-cell proliferation. To deplete arginine, MDSCs express high levels of arginase, which catalyzes the breakdown of arginine into urea and ornithine. Here, we developed a new hyperpolarized 13C probe, [6-13C]-arginine, to image arginase activity. We show that [6-13C]-arginine can be hyperpolarized, and hyperpolarized [13C]-urea production from [6-13C]-arginine is linearly correlated with arginase concentration in vitro. Furthermore we show that we can detect a statistically significant increase in hyperpolarized [13C]-urea production in MDSCs when compared to control bone marrow cells. This increase was associated with an increase in intracellular arginase concentration detected using a spectrophotometric assay. Hyperpolarized [6-13C]-arginine could therefore serve to image tumoral MDSC function and more broadly M2-like macrophages. PMID:27507680

  7. Structure of B sub 13 C sub 2

    SciTech Connect

    Bylander, D.M.; Kleinman, L. )

    1991-01-15

    By comparing calculated lattice constants with x-ray data as well as by comparison of calculated free energies, we find that the correct structure of B{sub 13}C{sub 2} is B{sub 12}(CBC) rather than B{sub 11}C(BBC), as had been suggested. We also show that B{sub 12}C{sub 3} is stable against 13B{sub 12}C{sub 3}{r arrow}12B{sub 13}C{sub 2}+15C as is B{sub 13}C{sub 2} against 3B{sub 13}C{sub 2}{r arrow}2B{sub 12}C{sub 3}+15B.

  8. Anomalous 13C enrichment in modern marine organic carbon

    USGS Publications Warehouse

    Arthur, M.A.; Dean, W.E.; Claypool, G.E.

    1985-01-01

    Marine organic carbon is heavier isotopically (13C enriched) than most land-plant or terrestrial organic C1. Accordingly, ??13C values of organic C in modern marine sediments are routinely interpreted in terms of the relative proportions of marine and terrestrial sources of the preserved organic matter2,3. When independent geochemical techniques are used to evaluate the source of organic matter in Cretaceous or older rocks, those rocks containing mostly marine organic C are found typically to have lighter (more-negative) ??13C values than rocks containing mostly terrestrial organic C. Here we conclude that marine photosynthesis in mid-Cretaceous and earlier oceans generally resulted in a greater fractionation of C isotopes and produced organic C having lighter ??13C values. Modern marine photosynthesis may be occurring under unusual geological conditions (higher oceanic primary production rates, lower PCO2) that limit dissolved CO2 availability and minimize carbon isotope fractionation4. ?? 1985 Nature Publishing Group.

  9. Dihydroflavanonols from Cedrus deodara, A (13)C NMR study.

    PubMed

    Agrawal, P K; Agarwal, S K; Rastogi, R P; Osterdahal, B G

    1981-09-01

    High resolution (13)C NMR study of taxifolin, cedeodarin, cedrin and their methyl ethers allowed unambiguous placement of the Me in 5,7-dihydroxyflavanonol nucleus, besides providing other valuable information on the substitution pattern in the molecule.

  10. Fecal /sup 13/C analysis for the detection and quantitation of intestinal malabsorption

    SciTech Connect

    Schoeller, D.A.; Klein, P.D.; MacLean, W.C. Jr.; Watkins, J.B.; Van Santen, E.

    1981-03-01

    The use of /sup 14/CO/sub 2/ breath tests and fecal analyses for the detection and quantitation of intestinal malabsorption has been extensively documented in adult subjects. The use of stable isotopes has extended the range of breath test applications to include pediatric and obstetric subjects. Here we report a fecal /sup 13/C analysis that can be used in conjunction with /sup 13/CO/sub 2/ breath tests. Twenty-four-hour fecal samples were collected before and after the administration of a labeled substrate. The samples were homogenized and combusted to CO/sub 2/, and the /sup 13/C abundance was determined by high-precision, differential isotope ratio mass spectrometry. The isotopic variation between successive 24 hr fecal samples was 0.6 per thousand (0.0006 atom percent). This variation limited the sensitivity of the fecal analysis to 13 ..mu..mol of /sup 13/C label per mole of fecal carbon. Simultaneous cholyglycine /sup 13/CO/sub 2/ breath tests and fecal assays were performed in five children. One child with bacterial overgrowth had an abnormal breath test and a normal fecal test. Of three children with ileal dysfunction, only one had an abnormal breath test, whereas the fecal test was abnormal in all three. Both the breath test and fecal test were abnormal for a child who had undergone an ileal resection. Both tests were normal for a child with ulcerative colitis.

  11. Pulsed polarization transfer for 13C NMR in solids

    NASA Astrophysics Data System (ADS)

    Bax, Ad; Szeverenyi, Nikolaus M.; Maciel, Gary E.

    A new pulsed polarization transfer experiment method is described for the polarization of 13C spins in a solid by magnetization transfer from protons. The method is directly analogous to the INEPT sequence for liquids introduced by Freeman and Morris. As polarization is transferred in PPT between individual 1H 13C pairs, rather than between spin reservoirs, different opportunities exist for structurally selective experiments. Results on p-diethoxybenzene and coronene are presented.

  12. Abundance anomaly of the 13C species of CCH

    NASA Astrophysics Data System (ADS)

    Sakai, N.; Saruwatari, O.; Sakai, T.; Takano, S.; Yamamoto, S.

    2010-03-01

    Aims: We have observed the N = 1-0 lines of CCH and its 13C isotopic species toward a cold dark cloud, TMC-1 and a star-forming region, L1527, to investigate the 13C abundances and formation pathways of CCH. Methods: The observations have been carried out with the IRAM 30 m telescope. Results: We have successfully detected the lines of 13CCH and C13CH toward the both sources and found a significant intensity difference between the two 13C isotopic species. The [C13CH] /[13CCH] abundance ratios are 1.6 ± 0.4 (3σ) and 1.6 ± 0.1 (3σ) for TMC-1 and L1527, respectively. The abundance difference between C13CH and 13CCH means that the two carbon atoms of CCH are not equivalent in the formation pathway. On the other hand, the [CCH]/[C13CH] and [CCH]/[13CCH] ratios are evaluated to be larger than 170 and 250 toward TMC-1, and to be larger than 80 and 135 toward L1527, respectively. Therefore, both of the 13C species are significantly diluted in comparison with the interstellar 12C/13C ratio of 60. The dilution is discussed in terms of a behavior of 13C in molecular clouds.

  13. Localized in vivo13C NMR spectroscopy of the brain

    PubMed Central

    Gruetter, Rolf; Adriany, Gregor; Choi, In-Young; Henry, Pierre-Gilles; Lei, Hongxia; Öz, Gülin

    2006-01-01

    Localized 13C NMR spectroscopy provides a new investigative tool for studying cerebral metabolism. The application of 13C NMR spectroscopy to living intact humans and animals presents the investigator with a number of unique challenges. This review provides in the first part a tutorial insight into the ingredients required for achieving a successful implementation of localized 13C NMR spectroscopy. The difficulties in establishing 13C NMR are the need for decoupling of the one-bond 13C–1H heteronuclear J coupling, the large chemical shift range, the low sensitivity and the need for localization of the signals. The methodological consequences of these technical problems are discussed, particularly with respect to (a) RF front-end considerations, (b) localization methods, (c) the low sensitivity, and (d) quantification methods. Lastly, some achievements of in vivo localized 13C NMR spectroscopy of the brain are reviewed, such as: (a) the measurement of brain glutamine synthesis and the feasibility of quantifying glutamatergic action in the brain; (b) the demonstration of significant anaplerotic fluxes in the brain; (c) the demonstration of a highly regulated malate-aspartate shuttle in brain energy metabolism and isotope flux; (d) quantification of neuronal and glial energy metabolism; and (e) brain glycogen metabolism in hypoglycemia in rats and humans. We conclude that the unique and novel insights provided by 13C NMR spectroscopy have opened many new research areas that are likely to improve the understanding of brain carbohydrate metabolism in health and disease. PMID:14679498

  14. 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

  15. Direct observation of glycogen synthesis in human muscle with sup 13 C NMR

    SciTech Connect

    Jue, T.; Rothman, D.L.; Shulman, G.I.; Tavitian, B.A.; DeFronzo, R.A.; Shulman, R.G. )

    1989-06-01

    On the basis of previous indirect measurements, skeletal muscle has been implicated as the major site of glucose uptake and it has been suggested that muscle glycogen formation is the dominant pathway. However, direct measurements of the rates of glycogen synthesis have not been possible by previous techniques. The authors have developed {sup 13}C NMR methods to measure directly the rate of human muscle glycogen formation from infused, isotopically labeled (1-{sup 13}C)glucose. They show that under conditions of imposed hyperglycemia and hyperinsulinemia, a majority of the infused glucose was converted to muscle glycogen in a normal man. This directly shows that muscle is the major site of glucose disposal under these conditions, and provides quantitation of the glucose flux to muscle glycogen.

  16. Landmarks in the application of 13C-magnetic resonance spectroscopy to studies of neuronal/glial relationships.

    PubMed

    Bachelard, H

    1998-01-01

    The development of the use of carbon isotopes as metabolic tracers is briefly described. 13C-labelled precursors (13CO2, 13CH4) first became available in 1940 and were studied in microorganisms, but their use was limited by very low enrichments and lack of suitable analytical equipment. More success was achieved with 11C and especially 14C, as these radioactive tracers did not need to be highly enriched. Although the stable 13C isotope can be used at a low percentage enrichment in mass spectrometry, its application to magnetic resonance spectroscopy (MRS) requires very highly enriched precursors, due to its low natural abundance and low sensitivity. Despite such limitations, however, the great advantage of 13C-MRS lies in its exquisite chemical specificity, in that labelling of different carbon atoms can be distinguished within the same molecule. Effective exploitation became feasible in the early 1970s with the advent of stable instruments, Fourier transform 13C-MRS, and the availability of highly enriched precursors. Reports of its use in brain research began to appear in the mid-1980s. The applications of 13C isotopomer analysis to research on neuronal/glial relationships are reviewed. The presence of neighbouring 13C-labelled atoms affects the appearance of the resonances (splitting due to C-C coupling), and so allows for unique quantification of rates through different and possibly competing pathways. Isotopomer patterns in resonances labelled from a combination of [1-13C]glucose and [1, 2-13C2]acetate have revealed aspects of neuronal/glial metabolic trafficking on depolarization and under hypoxic conditions in vitro. This approach has now been applied to in vivo studies on inhibition of glial metabolism using fluoroacetate. The results confirm the glial specificity of the toxin and demonstrate that it does not affect entry of acetate. When the glial TCA cycle is inhibited, the ability of the glia to participate in the glutamate/glutamine cycle remains

  17. Incorporation and Degradation of 14C and 3H-labeled Thymidine by Sugarcane Cells in Suspension Culture 12

    PubMed Central

    Lesley, Stanley M.; Maretzki, Andrew; Nickell, Louis G.

    1980-01-01

    Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-14C]thymidine is lost as 14CO2, but the label of [3H]methylthymidine is found in the cell as [3H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines. PMID:16661365

  18. Transport and imaging of brute-force (13)C hyperpolarization.

    PubMed

    Hirsch, Matthew L; Smith, Bryce A; Mattingly, Mark; Goloshevsky, Artem G; Rosay, Melanie; Kempf, James G

    2015-12-01

    We demonstrate transport of hyperpolarized frozen 1-(13)C pyruvic acid from its site of production to a nearby facility, where a time series of (13)C images was acquired from the aqueous dissolution product. Transportability is tied to the hyperpolarization (HP) method we employ, which omits radical electron species used in other approaches that would otherwise relax away the HP before reaching the imaging center. In particular, we attained (13)C HP by 'brute-force', i.e., using only low temperature and high-field (e.g., T<∼2K and B∼14T) to pre-polarize protons to a large Boltzmann value (∼0.4% (1)H polarization). After polarizing the neat, frozen sample, ejection quickly (<1s) passed it through a low field (B<100G) to establish the (1)H pre-polarization spin temperature on (13)C via the process known as low-field thermal mixing (yielding ∼0.1% (13)C polarization). By avoiding polarization agents (a.k.a. relaxation agents) that are needed to hyperpolarize by the competing method of dissolution dynamic nuclear polarization (d-DNP), the (13)C relaxation time was sufficient to transport the sample for ∼10min before finally dissolving in warm water and obtaining a (13)C image of the hyperpolarized, dilute, aqueous product (∼0.01% (13)C polarization, a >100-fold gain over thermal signals in the 1T scanner). An annealing step, prior to polarizing the sample, was also key for increasing T1∼30-fold during transport. In that time, HP was maintained using only modest cryogenics and field (T∼60K and B=1.3T), for T1((13)C) near 5min. Much greater time and distance (with much smaller losses) may be covered using more-complete annealing and only slight improvements on transport conditions (e.g., yielding T1∼5h at 30K, 2T), whereas even intercity transfer is possible (T1>20h) at reasonable conditions of 6K and 2T. Finally, it is possible to increase the overall enhancement near d-DNP levels (i.e., 10(2)-fold more) by polarizing below 100mK, where nanoparticle

  19. Transport and imaging of brute-force 13C hyperpolarization

    NASA Astrophysics Data System (ADS)

    Hirsch, Matthew L.; Smith, Bryce A.; Mattingly, Mark; Goloshevsky, Artem G.; Rosay, Melanie; Kempf, James G.

    2015-12-01

    We demonstrate transport of hyperpolarized frozen 1-13C pyruvic acid from its site of production to a nearby facility, where a time series of 13C images was acquired from the aqueous dissolution product. Transportability is tied to the hyperpolarization (HP) method we employ, which omits radical electron species used in other approaches that would otherwise relax away the HP before reaching the imaging center. In particular, we attained 13C HP by 'brute-force', i.e., using only low temperature and high-field (e.g., T < ∼2 K and B ∼ 14 T) to pre-polarize protons to a large Boltzmann value (∼0.4% 1H polarization). After polarizing the neat, frozen sample, ejection quickly (<1 s) passed it through a low field (B < 100 G) to establish the 1H pre-polarization spin temperature on 13C via the process known as low-field thermal mixing (yielding ∼0.1% 13C polarization). By avoiding polarization agents (a.k.a. relaxation agents) that are needed to hyperpolarize by the competing method of dissolution dynamic nuclear polarization (d-DNP), the 13C relaxation time was sufficient to transport the sample for ∼10 min before finally dissolving in warm water and obtaining a 13C image of the hyperpolarized, dilute, aqueous product (∼0.01% 13C polarization, a >100-fold gain over thermal signals in the 1 T scanner). An annealing step, prior to polarizing the sample, was also key for increasing T1 ∼ 30-fold during transport. In that time, HP was maintained using only modest cryogenics and field (T ∼ 60 K and B = 1.3 T), for T1(13C) near 5 min. Much greater time and distance (with much smaller losses) may be covered using more-complete annealing and only slight improvements on transport conditions (e.g., yielding T1 ∼ 5 h at 30 K, 2 T), whereas even intercity transfer is possible (T1 > 20 h) at reasonable conditions of 6 K and 2 T. Finally, it is possible to increase the overall enhancement near d-DNP levels (i.e., 102-fold more) by polarizing below 100 mK, where

  20. Unraveling the 13C NMR Chemical Shifts in Single-Walled Carbon Nanotubes: Dependence on Diameter and Electronic Structure

    SciTech Connect

    Engtrakul, C.; Irurzun, V. M.; Gjersing, E. L.; Holt, J. M.; Larsen, B. A.; Resasco, D. E.; Blackburn, J. L.

    2012-03-14

    The atomic specificity afforded by nuclear magnetic resonance (NMR) spectroscopy could enable detailed mechanistic information about single-walled carbon nanotube (SWCNT) functionalization as well as the noncovalent molecular interactions that dictate ground-state charge transfer and separation by electronic structure and diameter. However, to date, the polydispersity present in as-synthesized SWCNT populations has obscured the dependence of the SWCNT {sup 13}C chemical shift on intrinsic parameters such as diameter and electronic structure, meaning that no information is gleaned for specific SWCNTs with unique chiral indices. In this article, we utilize a combination of {sup 13}C labeling and density gradient ultracentrifugation (DGU) to produce an array of {sup 13}C-labeled SWCNT populations with varying diameter, electronic structure, and chiral angle. We find that the SWCNT isotropic {sup 13}C chemical shift decreases systematically with increasing diameter for semiconducting SWCNTs, in agreement with recent theoretical predictions that have heretofore gone unaddressed. Furthermore, we find that the {sup 13}C chemical shifts for small diameter metallic and semiconducting SWCNTs differ significantly, and that the full-width of the isotropic peak for metallic SWCNTs is much larger than that of semiconducting nanotubes, irrespective of diameter.

  1. A 13C-NMR study of azacryptand complexes.

    PubMed

    Wild, Aljoscha A C; Fennell, Kevin; Morgan, Grace G; Hewage, Chandralal M; Malthouse, J Paul G

    2014-09-28

    An azacryptand has been solubilised in aqueous media containing 50% (v/v) dimethyl sulphoxide. (13)C-NMR has been used to determine how the azacryptand is affected by zinc binding at pH 10. Using (13)C-NMR and (13)C-enriched bicarbonate we have been able to observe the formation of 4 different carbamate derivatives of the azacryptand at pH 10. The azacryptand was shown to solubilise zinc or cadmium at alkaline pHs. Two moles of zinc are bound per mole of azacryptand and this complex binds 1 mole of carbonate. By replacing the zinc with cadmium-113 we have shown that the (13)C-NMR signal of the (13)C-enriched carbon of the bound carbonate is split into two triplets at 2.2 °C. This shows that two cadmium complexes are formed and in each of these complexes the carbonate group is bound by two magnetically equivalent metal ions. It also demonstrates that these cadmium complexes are not in fast exchange. From temperature studies we show that in the zinc complexes both complexes are in fast exchange with each other but are in slow exchange with free bicarbonate. HOESY is used to determine the position of the carbonate carbon in the complex. The solution and crystal structures of the zinc-carbonate-azacryptand complexes are compared. PMID:25091182

  2. Natural isotope correction of MS/MS measurements for metabolomics and (13)C fluxomics.

    PubMed

    Niedenführ, Sebastian; ten Pierick, Angela; van Dam, Patricia T N; Suarez-Mendez, Camilo A; Nöh, Katharina; Wahl, S Aljoscha

    2016-05-01

    Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of (13)C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full use of LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; (13)C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation.

  3. Natural isotope correction of MS/MS measurements for metabolomics and (13)C fluxomics.

    PubMed

    Niedenführ, Sebastian; ten Pierick, Angela; van Dam, Patricia T N; Suarez-Mendez, Camilo A; Nöh, Katharina; Wahl, S Aljoscha

    2016-05-01

    Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of (13)C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full use of LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; (13)C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation. PMID:26479486

  4. (13) C-TmDOTA as versatile thermometer compound for solid-state NMR of hydrated lipid bilayer membranes.

    PubMed

    Umegawa, Yuichi; Tanaka, Yuya; Nobuaki, Matsumori; Murata, Michio

    2016-03-01

    Recent advances in solid-state nuclear magnetic resonance (NMR) techniques, such as magic angle spinning and high-power decoupling, have dramatically increased the sensitivity and resolution of NMR. However, these NMR techniques generate extra heat, causing a temperature difference between the sample in the rotor and the variable temperature gas. This extra heating is a particularly crucial problem for hydrated lipid membrane samples. Thus, to develop an NMR thermometer that is suitable for hydrated lipid samples, thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA) was synthesized and labeled with (13) C (i.e., (13) C-TmDOTA) to increase the NMR sensitivity. The complex was mixed with a hydrated lipid membrane, and the system was subjected to solid-state NMR and differential scanning calorimetric analyses. The physical properties of the lipid bilayer and the quality of the NMR spectra of the membrane were negligibly affected by the presence of (13) C-TmDOTA, and the (13) C chemical shift of the complex exhibited a large-temperature dependence. The results demonstrated that (13) C-TmDOTA could be successfully used as a thermometer to accurately monitor temperature changes induced by (1) H decoupling pulses and/or by magic angle spinning and the temperature distribution of the sample inside the rotor. Thus, (13) C-TmDOTA was shown to be a versatile thermometer for hydrated lipid assemblies. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26460094

  5. (13) C-TmDOTA as versatile thermometer compound for solid-state NMR of hydrated lipid bilayer membranes.

    PubMed

    Umegawa, Yuichi; Tanaka, Yuya; Nobuaki, Matsumori; Murata, Michio

    2016-03-01

    Recent advances in solid-state nuclear magnetic resonance (NMR) techniques, such as magic angle spinning and high-power decoupling, have dramatically increased the sensitivity and resolution of NMR. However, these NMR techniques generate extra heat, causing a temperature difference between the sample in the rotor and the variable temperature gas. This extra heating is a particularly crucial problem for hydrated lipid membrane samples. Thus, to develop an NMR thermometer that is suitable for hydrated lipid samples, thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA) was synthesized and labeled with (13) C (i.e., (13) C-TmDOTA) to increase the NMR sensitivity. The complex was mixed with a hydrated lipid membrane, and the system was subjected to solid-state NMR and differential scanning calorimetric analyses. The physical properties of the lipid bilayer and the quality of the NMR spectra of the membrane were negligibly affected by the presence of (13) C-TmDOTA, and the (13) C chemical shift of the complex exhibited a large-temperature dependence. The results demonstrated that (13) C-TmDOTA could be successfully used as a thermometer to accurately monitor temperature changes induced by (1) H decoupling pulses and/or by magic angle spinning and the temperature distribution of the sample inside the rotor. Thus, (13) C-TmDOTA was shown to be a versatile thermometer for hydrated lipid assemblies. Copyright © 2015 John Wiley & Sons, Ltd.

  6. Quantification of dung carbon incorporation in a temperate grassland soil following spring application using bulk stable carbon isotope determinations.

    PubMed

    Dungait, J A J; Bol, R; Evershed, R P

    2005-03-01

    Herbivore dung constitutes a substantial input of C to temperate grassland soils, and its fate must be determined in order to fully understand nutrient cycling in this ecosystem. This experiment used changes in bulk delta13C values of the 0-1 cm and 1-5 cm soil horizons of a dung-treated temperate grassland soil to approximate percentage applied dung C incorporation over 372 days. Natural abundance 13C-labelled C4 dung (delta13C - 12.6%) and C3 dung (delta13C - 31.3% were produced in a monitored diet switch from ryegrass silage (delta13C - 30.1%) to maize silage (delta13C - 11.6%). The dung was applied to a C3 grassland (delta13C 0-1 cm - 29.9%, 1-5 cm - 30.6%), and dung remains and soil cores from beneath the treatments were sampled at intervals. delta13C values were used to estimate a maximum of 12% applied dung C incorporation in the top 5 cm of the soil after 112 days, which declined to around 8% at the end of the experiment. A significant increase in percentage applied dung C was observed in the top 1 cm of soil, compared with the 1-5 cm horizon, after a substantial rain event after 30 days. However, results of forage fibre analyses of the two dung types revealed significant differences in composition which may affect subsequent calculations of percentage dung incorporation based on bulk delta13C values.

  7. Spectral density mapping at multiple magnetic fields suitable for 13C NMR relaxation studies

    NASA Astrophysics Data System (ADS)

    Kadeřávek, Pavel; Zapletal, Vojtěch; Fiala, Radovan; Srb, Pavel; Padrta, Petr; Přecechtělová, Jana Pavlíková; Šoltésová, Mária; Kowalewski, Jozef; Widmalm, Göran; Chmelík, Josef; Sklenář, Vladimír; Žídek, Lukáš

    2016-05-01

    Standard spectral density mapping protocols, well suited for the analysis of 15N relaxation rates, introduce significant systematic errors when applied to 13C relaxation data, especially if the dynamics is dominated by motions with short correlation times (small molecules, dynamic residues of macromolecules). A possibility to improve the accuracy by employing cross-correlated relaxation rates and on measurements taken at several magnetic fields has been examined. A suite of protocols for analyzing such data has been developed and their performance tested. Applicability of the proposed protocols is documented in two case studies, spectral density mapping of a uniformly labeled RNA hairpin and of a selectively labeled disaccharide exhibiting highly anisotropic tumbling. Combination of auto- and cross-correlated relaxation data acquired at three magnetic fields was applied in the former case in order to separate effects of fast motions and conformational or chemical exchange. An approach using auto-correlated relaxation rates acquired at five magnetic fields, applicable to anisotropically moving molecules, was used in the latter case. The results were compared with a more advanced analysis of data obtained by interpolation of auto-correlated relaxation rates measured at seven magnetic fields, and with the spectral density mapping of cross-correlated relaxation rates. The results showed that sufficiently accurate values of auto- and cross-correlated spectral density functions at zero and 13C frequencies can be obtained from data acquired at three magnetic fields for uniformly 13C -labeled molecules with a moderate anisotropy of the rotational diffusion tensor. Analysis of auto-correlated relaxation rates at five magnetic fields represents an alternative for molecules undergoing highly anisotropic motions.

  8. {sup 13}C relaxation in an RNA hairpin

    SciTech Connect

    King, G.C. |; Akratos, C.; Xi, Z.; Michnica, M.J.

    1994-12-01

    This initial survey of {sup 13}C relaxation in the {triangle}TAR RNA element has generated a number of interesting results that should prove generally useful for future studies. The most readily comparable study in the literature monitored {sup 13}C relaxation of the methyl groups from unusual bases in tRNA{sup Phe}. The study, which used T{sub 1} and NOE data only, reported order parameters for the methyl group axis that ranged between 0.51 and 0.97-a range similar to that observed here. However, they reported a breakdown of the standard order parameter analysis at higher (118-MHz {sup 13}C) frequencies, which should serve to emphasize the need for a thorough exploration of suitable motional models.

  9. The economical synthesis of [2'-(13)C, 1,3-(15)N2]uridine; preliminary conformational studies by solid state NMR.

    PubMed

    Patching, Simon G; Middleton, David A; Henderson, Peter J F; Herbert, Richard B

    2003-06-21

    The synthesis of [2'-(13)C, 1,3-(15)N2]uridine 11 was achieved as follows. An epimeric mixture of D-[1-(13)C]ribose 3 and D-[1-(13)C]arabinose 4 was obtained in excellent yield by condensation of K13CN with D-erythrose 2 using a modification of the Kiliani-Fischer synthesis. Efficient separation of the two aldose epimers was pivotally achieved by a novel ion-exchange (Sm3+) chromatography method. D-[2-(13)C]Ribose 5 was obtained from D-[1-(13)C]arabinose 4 using a Ni(II) diamine complex (nickel chloride plus TEMED). Combination of these procedures in a general cycling manner can lead to the very efficient preparation of specifically labelled 13C-monosaccharides of particular chirality. 15N-labelling was introduced in the preparation of [2'-(13)C, 1,3-(15)N2]uridine 11 via [15N2]urea. Cross polarisation magic angle spinning (CP-MAS) solid-state NMR experiments using rotational echo double resonance (REDOR) were carried out on crystals of the labelled uridine to show that the inter-atomic distance between C-2' and N-1 is closely similar to that calculated from X-ray crystallographic data. The REDOR method will be used now to determine the conformation of bound substrates in the bacterial nucleoside transporters NupC and NupG.

  10. Screening and Selection of High Carotenoid Producing in Vitro Tomato Cell Culture Lines for [13C]-Carotenoid Production

    PubMed Central

    Engelmann, Nancy J.; Campbell, Jessica K.; Rogers, Randy B.; Rupassara, S. Indumathie; Garlick, Peter J.; Lila, Mary Ann; Erdman, John W.

    2011-01-01

    Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)-triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-labeling. Different Solanum lycopersicum allelic variants for high lycopene and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture, and cell suspension cultures of the hp-1 line were chosen for isotopic labeling. When grown with [U]-13C-glucose and treated with CPTA, hp-1 suspensions yielded highly enriched 13C-lycopene with 45% of lycopene in the M+40 form and 88% in the M+35 to M+40 isotopomer range. To the authors’ knowledge this is the first report of highly enriched 13C-carotenoid production from in vitro plant cell culture. PMID:20731353

  11. Preparation of base-modified nucleosides suitable for non-radioactive label attachment and their incorporation into synthetic oligodeoxyribonucleotides.

    PubMed Central

    Haralambidis, J; Chai, M; Tregear, G W

    1987-01-01

    A very mild and efficient procedure has been developed for the preparation of C-5 substituted deoxyuridines. The substituent carries a masked primary aliphatic amino group. These compounds are readily converted into their phosphoramidites and can be used to prepare oligonucleotides carrying one or more aliphatic amino groups. Fluorescein isothiocyanate coupled to these compounds gives oligonucleotide probes carrying multiple fluorescein labels. These compounds have a free 5'-hydroxy group enabling additional 5'- end radioactive labelling for evaluation of their hybridization characteristics. It was found that oligonucleotides carrying a long (11 atom) linker arm to the fluorescein hybridize more efficiently to mRNA than those carrying a short (4 atom) arm. The long linker arm derivatives are comparable to underivatized oligonucleotides in hybridizing to mRNA. Images PMID:3110740

  12. Measuring doubly 13C-substituted ethane by mass spectrometry

    NASA Astrophysics Data System (ADS)

    Clog, M.; Ling, C.; Eiler, J. M.

    2012-12-01

    Ethane (C2H6) is present in non-negligible amounts in most natural gas reservoirs and is used to produce ethylene for petrochemical industries. It is one of the by-products of lipid metabolism and is the arguably simplest molecule that can manifest multiple 13C substitutions. There are several plausible controls on the relative abundances of 13C2H6 in natural gases: thermodynamically controlled homogeneous isotope exchange reactions analogous to those behind carbonate clumped isotope thermometry; inheritance from larger biomolecules that under thermal degradation to produce natural gas; mixing of natural gases that differ markedly in bulk isotopic composition; or combinations of these and/or other, less expected fractionations. There is little basis for predicting which of these will dominate in natural samples. Here, we focus on an analytical techniques that will provide the avenue for exploring these phenomena. The method is based on high-resolution gas source isotope ratio mass spectrometry, using the Thermo 253-Ultra (a new prototype mass spectrometer). This instrument achieves the mass resolution (M/Δ M) up to 27,000, permitting separation of the isobaric interferences of potential contaminants and isotopologues of an analtye or its fragments which share a cardinal mass. We present techniques to analyze several isotopologues of molecular and fragment ions of C2H6. The critical isobaric separations for our purposes include: discrimination of 13C2H6 from 13C12CDH5 at mass 32 and separation of the 13CH3 fragment from 12CH4 at mass 16, both requiring at least a mass resolution of 20000 to make an adequate measurement. Other obvious interferences are either cleanly separated (e.g., O2, O) or accounted for by peak-stripping (CH3OH on mass 32 and NH2 on mass 16). We focus on a set of measurements which constrain: the doubly-substituted isotopologue, 13C2H6, and the 13CH3/12CH3 ratio of the methyl fragment, which constrains the bulk δ 13C. Similar methods can be

  13. Optimized [1-13C]glucose infusion protocol for 13C magnetic resonance spectroscopy at 3 Tesla of human brain glucose metabolism under euglycemic and hypoglycemic conditions

    PubMed Central

    van de Ven, Kim C.C.; van der Graaf, Marinette; Tack, Cees J.J.; Klomp, Dennis W.J.; Heerschap, Arend; de Galan, Bastiaan E.

    2009-01-01

    The effect of insulin-induced hypoglycemia on cerebral glucose metabolism is largely unknown. 13C MRS is a unique tool to study cerebral glucose metabolism, but the concurrent requirement for [1-13C]glucose administration limits its use under hypoglycemic conditions. To facilitate 13C MRS data analysis we designed separate [1-13C]glucose infusion protocols for hyperinsulinemic euglycemic and hypoglycemic clamps in such a way that plasma isotopic enrichment of glucose was stable and comparable under both glycemic conditions. 13C MR spectra were acquired with optimized 13C MRS measurement techniques to obtain high quality 13C MR spectra with these protocols. PMID:19913052

  14. /sup 13/C nuclear magnetic resonance studies of the biosynthesis by Microbacterium ammoniaphilum of L-glutamate selectively enriched with carbon-13

    SciTech Connect

    Walker, T.E.; Han, C.H.; Kollman, V.H.; London, R.E.; Matwiyoff, N.A.

    1982-02-10

    /sup 13/C NMR of isotopically enriched metabolites has been used to study the metabolism of Microbacterium ammoniaphilum, a bacterium which excretes large quantities of L-glutamic acid into the medium. Biosynthesis from 90% (1-/sup 13/C) glucose results in relatively high specificity of the label, with (2,4-/sup 13/C/sub 2/) glutamate as the major product. The predominant biosynthetic pathway for synthesis of glutamate from glucose was determined to be the Embden Meyerhof glycolytic pathway followed by P-enolpyruvate carboxylase and the first third of the Krebs cycle. Different metabolic pathways are associated with different correlations in the enrichment of the carbons, reflected in the spectrum as different /sup 13/C-/sup 13/C scalar multiplet intensities. Hence, intensity and /sup 13/C-/sup 13/C multiplet analysis allows quantitation of the pathways involved. Although blockage of the Krebs cycle at the ..cap alpha..-ketoglutarate dehydrogenase step is the basis for the accumulation of glutamate, significant Krebs cycle activity was found in glucose grown cells, and extensive Krebs cycle activity in cells metabolizing (1-/sup 13/C) acetate. In addition to the observation of the expected metabolites, the disaccharide ..cap alpha..,..cap alpha..-trehalose and ..cap alpha..,..beta..-glucosylamine were identified from the /sup 13/C NMR spectra.

  15. SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis

    PubMed Central

    Kogadeeva, Maria

    2016-01-01

    Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses. PMID:27626798

  16. SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis.

    PubMed

    Kogadeeva, Maria; Zamboni, Nicola

    2016-09-01

    Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses. PMID:27626798

  17. Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin- or fluorescein-labeled dUTP

    SciTech Connect

    Yang, Gang; Olson, J.C.; Pu, R.; Vyas, G.N.

    1995-10-01

    Serological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type-1 (HrV-1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein-12-dUTP (fluo-dUTP) or digoxigenin-11-dUTP (dig-dUTP) into the PCR-amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin-coated beads which were finally analyzed in a flow cytometer by (1) direct detection of the fluorescence intensity of the amplicons incorporating fluo-dUTP and (2) immunodetection of the amplicons incorporating dig-dUTP by anti-digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV-1 proviral DNA sequences, the immunodetection of dig-dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12-14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV-1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig-dUTP incorporation in amplicons, hybridization with a pair of sense-antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR-diagnosis of HIV-1 infection. 21 refs., 2 figs., 4 tabs.

  18. Neutron Backgrounds: 13C({alpha}, n) etc

    SciTech Connect

    Mitsui, Tadao

    2005-09-08

    13C({alpha}, n) reaction is the main neutron source in an underground large-volume liquid-scintillator detector KamLAND. {alpha} sources, targets, cross sections, and neutron transport are studied to estimate the backgrounds of v-bare signal.

  19. Spectral editing for in vivo 13C magnetic resonance spectroscopy

    NASA Astrophysics Data System (ADS)

    Xiang, Yun; Shen, Jun

    2012-01-01

    In vivo detection of carboxylic/amide carbons is a promising technique for studying cerebral metabolism and neurotransmission due to the very low RF power required for proton decoupling. In the carboxylic/amide region, however, there is severe spectral overlap between acetate C1 and glutamate C5, complicating studies that use acetate as an astroglia-specific substrate. There are no known in vivo MRS techniques that can spectrally resolve acetate C1 and glutamate C5 singlets. In this study, we propose to spectrally separate acetate C1 and glutamate C5 by a two-step J-editing technique after introducing homonuclear 13C- 13C scalar coupling between carboxylic/amide carbons and aliphatic carbons. By infusing [1,2- 13C 2]acetate instead of [1- 13C]acetate the acetate doublet can be spectrally edited because of the large separation between acetate C2 and glutamate C4 in the aliphatic region. This technique can be applied to studying acetate transport and metabolism in brain in the carboxylic/amide region without spectral interference.

  20. Stable isotope-labeled RNA phosphoramidites to facilitate dynamics by NMR.

    PubMed

    Wunderlich, Christoph H; Juen, Michael A; LeBlanc, Regan M; Longhini, Andrew P; Dayie, T Kwaku; Kreutz, Christoph

    2015-01-01

    Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes, methods to synthesize these RNAs for biophysical studies are much needed. Here, we showcase the applicability of 6-(13)C-pyrimidine phosphoramidites to introduce isolated (13)C-(1)H spin pairs into RNAs up to 40 nucleotides long. The method allows the incorporation of 6-(13)C-uridine and -cytidine residues at any desired position within a target RNA. By site-specific positioning of the (13)C-label using RNA solid phase synthesis, these stable isotope-labeling patterns are especially well suited to resolve resonance assignment ambiguities. Of even greater importance, the labeling pattern affords accurate quantification of important functional transitions of biologically relevant RNAs (e.g., riboswitch aptamer domains, viral RNAs, or ribozymes) in the μs- to ms time regime and beyond without complications of one bond carbon scalar couplings. We outline the chemical synthesis of the 6-(13)C-pyrimidine building blocks and their use in RNA solid phase synthesis and demonstrate their utility in Carr Purcell Meiboom Gill relaxation dispersion, ZZ exchange, and chemical exchange saturation transfer NMR experiments. PMID:26577742

  1. Does the Shuram δ13C excursion record Ediacaran oxygenation?

    NASA Astrophysics Data System (ADS)

    Husson, J. M.; Maloof, A. C.; Schoene, B.; Higgins, J. A.

    2013-12-01

    The most negative carbon isotope excursion in Earth history is found in carbonate rocks of the Ediacaran Period (635-542 Ma). Known colloquially as the the 'Shuram' excursion, workers have long noted its tantalizing, broad concordance with the rise of abundant macro-scale fossils in the rock record, variously interpreted as animals, giant protists, macro-algae and lichen, and known as the 'Ediacaran Biota.' Thus, the Shuram excursion has been interpreted by many in the context of a dramatically changing redox state of the Ediacaran oceans - e.g., a result of methane cycling in a low O2 atmosphere, the final destruction of a large pool of recalcitrant dissolved organic carbon (DOC), and the step-wise oxidation of the Ediacaran oceans. More recently, diagenetic interpretations of the Shuram excursion - e.g. sedimentary in-growth of very δ13C depleted authigenic carbonates, meteoric alteration of Ediacaran carbonates, late-stage burial diagenesis - have challenged the various Ediacaran redox models. A rigorous geologic context is required to discriminate between these explanatory models, and determine whether the Shuram excursion can be used to evaluate terminal Neoproterozoic oxygenation. Here, we present chemo-stratigraphic data (δ13C, δ18O, δ44/42Ca and redox sensitive trace element abundances) from 12 measured sections of the Ediacaran-aged Wonoka Formation (Fm.) of South Australia that require a syn-depositional age for the extraordinary range of δ13C values (-12 to +4‰) observed in the formation. In some locations, the Wonoka Fm. is ~700 meters (m) of mixed shelf limestones and siliclastics that record the full 16 ‰ δ13C excursion in a remarkably consistent fashion across 100s of square kilometers of basin area. Fabric-altering diagenesis, where present, occurs at the sub-meter vertical scale, only results in sub-permil offsets in δ13C and cannot be used to explain the full δ13C excursion. In other places, the Wonoka Fm. is host to deep (1 km

  2. Incorporation of hydrogen atoms from deuterated water and stereospecifically deuterium-labeled nicotin amide nucleotides into fatty acids with the Escherichia coli fatty acid synthetase system.

    PubMed

    Saito, K; Kawaguchi, A; Okuda, S; Seyama, Y; Yamakawa, T

    1980-05-28

    The mechanism of hydrogen incorporation into fatty acids was investigated with intact Escherichia coli cells, a crude enzyme preparation and purified reductases of fatty acid synthetase system. The distributions of deuterium atoms incorporated into fatty acids from 2H2O and stereospecifically deuterium-labeled NADPH or NADH were determined by mass spectrometry. When E. coli was grown in 2H2O, almost every hydrogen atom of cellular fatty acids was incorporated from the medium. When fatty acids were synthesized from acetyl-CoA, malonyl-CoA and NADPH in the presence of a crude enzyme preparation of either E. coli or Bacillus subtilis, almost every hydrogen atom was also incorporated from the medium. In contrast to these results, purified beta-ketoacyl acyl carrier reductase directly transferred the HB hydrogen of NADPH to beta-ketoacyl acyl carrier protein, and purified enoyl acyl carrier protein reductase also transferred the HB hydrogen of NADPH and NADH directly to enoyl acyl carrier protein. In the crude enzyme preparation of E. coli, we found high activities which exchanged the HB hydrogen of NADPH with the deuterium of 2h2o. the conflicting results of the origin of hydrogen atoms of fatty acids mentioned above are explained by the presence of enzymes, which catalyzed the rapid exchange of NADPH with the deterium of 2H2O prior to the reaction of fatty acid synthetase. PMID:6990992

  3. Optical hyperpolarization of 13C nuclear spins in nanodiamond ensembles

    NASA Astrophysics Data System (ADS)

    Chen, Q.; Schwarz, I.; Jelezko, F.; Retzker, A.; Plenio, M. B.

    2015-11-01

    Dynamical nuclear polarization holds the key for orders of magnitude enhancements of nuclear magnetic resonance signals which, in turn, would enable a wide range of novel applications in biomedical sciences. However, current implementations of DNP require cryogenic temperatures and long times for achieving high polarization. Here we propose and analyze in detail protocols that can achieve rapid hyperpolarization of 13C nuclear spins in randomly oriented ensembles of nanodiamonds at room temperature. Our protocols exploit a combination of optical polarization of electron spins in nitrogen-vacancy centers and the transfer of this polarization to 13C nuclei by means of microwave control to overcome the severe challenges that are posed by the random orientation of the nanodiamonds and their nitrogen-vacancy centers. Specifically, these random orientations result in exceedingly large energy variations of the electron spin levels that render the polarization and coherent control of the nitrogen-vacancy center electron spins as well as the control of their coherent interaction with the surrounding 13C nuclear spins highly inefficient. We address these challenges by a combination of an off-resonant microwave double resonance scheme in conjunction with a realization of the integrated solid effect which, together with adiabatic rotations of external magnetic fields or rotations of nanodiamonds, leads to a protocol that achieves high levels of hyperpolarization of the entire nuclear-spin bath in a randomly oriented ensemble of nanodiamonds even at room temperature. This hyperpolarization together with the long nuclear-spin polarization lifetimes in nanodiamonds and the relatively high density of 13C nuclei has the potential to result in a major signal enhancement in 13C nuclear magnetic resonance imaging and suggests functionalized and hyperpolarized nanodiamonds as a unique probe for molecular imaging both in vitro and in vivo.

  4. Reflections on the application of 13C-MRS to research on brain metabolism.

    PubMed

    Morris, Peter; Bachelard, Herman

    2003-01-01

    The power of (13)C-MRS lies in its unique chemical specificity, enabling detection and quantification of metabolic intermediates which would not be so readily monitored using conventional radiochemical techniques. Examples from animal studies, by examination of tissue extracts from the whole brain, brain slices and cultured cells, include observation of intermediates such as citrate and triose phosphates which have yielded novel information on neuronal/glial relationships. The use of (13)C-labelled acetate as a specific precursor for glial metabolism provided evidence in support of the view that some of the GABA produced in the brain is derived from glial glutamine. Such studies have also provided direct evidence on the contribution of anaplerotic pathways to intermediary metabolism. Analogous studies are now being performed on the human brain, where (13)C-acetate is used to quantitate the overall contribution of glial cells to intermediary metabolism, and use of (13)C-glucose enables direct calculation of rates of flux through the TCA (F(TCA)) and of the glutamate-glutamine cycle (F(CYC)), leading to the conclusion that the rate of glial recycling of glutamate accounts for some 50% of F(TCA). The rate of 0.74 micromol min(-1) g(-1) for F(TCA) is compatible with PET rates of CMRglc of 0.3-0.4 micromol min(-1) g(-1) (since each glucose molecule yields two molecules of pyruvate entering the TCA). Our brain activation studies showed a 60% increase in F(TCA), which is very similar to the increases in CBF and in CMRglc observed in PET activation studies.

  5. Changes in the 14C-Labeled Cell Wall Components with Chase Time after Incorporation of UDP[14C]Glucose by Intact Cotton Fibers 1

    PubMed Central

    Dugger, W. M.; Palmer, Raymond L.

    1988-01-01

    Intact, in vitro-grown cotton fibers will incorporate [14C]glucose from externally supplied UDP[14C]glucose into a variety of cell wall components including cellulose; this labeled fraction will continue to increase up to 4 hours chase time. In the fraction soluble in hot water there was no significant change in total label; however, the largest fraction after the 30 minute pulse with UDP[14C]glucose was chloroform-methanol soluble (70%) and showed a significant decrease with chase. The lipids that make up about 85% of this fraction were identified by TLC as steryl glucosides, acylated steryl glucosides, and glucosyl-phosphoryl-polyprenol. Following the pulse, the loss of label from acylated steryl glucosides and glucosylphophoryl-polyprenol was almost complete within 2 hours of chase; steryl glucosides made up about 85% of the fraction at that chase time. The total loss in the lipid fraction (about 100 picomoles per milligram dry weight of fiber) with chase times of 4 hours approximates the total gain in the total glucans. PMID:16666066

  6. Convenient synthesis of nickel (5,7,12,14,19,21,26,28- sup 13 C sub 8 )phthalocyanine

    SciTech Connect

    Barrett, A.G.M.; Broderick, W.E.; Hoffman, B.M.; Velazquez, C.S. )

    1989-06-23

    Metallophthalocyanines are convenient precursors for diverse low-dimensional electrical conductors. Recently we wished to prepare large quantities of nickel (5,7,12,14,19,21,26,28-{sup 13}C{sub 8})phthalocyanine (4) with high isotopic enrichment. Previously macrocycle 4 had been prepared at five times natural abundance by the cyclization of 1,2-dicyanobenzene (3). The partially labeled 1,2-dicyanobenzene (3) in turn was prepared from {sup 13}C-enriched potassium cyanide through the use of copper(I) cyanide. However, we were reluctant to employ this methodology to achieve greater enrichment because of the high cost of 99% potassium ({sup 13}C)cyanide and low overall yield of the process. Herein we report an efficient method to prepare 4 using (arene)tricarbonylchromium chemistry.

  7. Fossil chironomid d13C as a new proxy for past methanogenic contribution to benthic food-webs in lakes?

    NASA Astrophysics Data System (ADS)

    van Hardenbroek, M.; Heiri, O. M.; Grey, J.; Bodelier, P. L. E.; Lotter, A. F.

    2009-04-01

    Lake sediments are an important source of atmospheric methane. Methanogenic archaea in lake sediments produce 13C-depleted methane that is partly released to the water column and the atmosphere. Another part is utilized by methane oxidizing bacteria (MOB) that are an important food source for deposit-feeding chironomid larvae (Diptera: Chironomidae). If methane-derived carbon is a significant component of the chironomid diet this will lead to strongly negative d13C in the tissue and exoskeleton of chironomid larvae. Chironomid cuticles, especially the strongly sclerotized head capsules, are well preserved as fossils in lake sediments. If the relationship between modern methane fluxes in lakes and chironomid d13C can be established this would therefore provide an approach for estimating past methane fluxes based on d13C of fossil chironomid remains. Using culturing experiments we show that the stable carbon isotope signature of MOB and other food sources can be traced in chironomid muscle tissue as well as in the fossilizing exoskeleton. In addition we measured d13C in chironomid larval head capsules and other invertebrate remains from a range of surface and downcore sediment samples. Small intra-specific variability (-27.1 ± 0.08 permille) was measured in replicate samples of chironomid head capsules of Corynocera ambigua (n=7). d13C of chironomid head capsules from a several different taxa ranged from -28.0 to -25.8 permille, but in some instances we observed d13C values as low as -36.9 to -31.5 permille, suggesting that carbon from MOB can be successfully traced in fossil and subfossil chironomid remains. Our results demonstrate that the stable carbon isotope signature of MOB is incorporated into chironomid head capsules. Future research will focus on quantifying the relationship between methane fluxes, MOB, and head capsule d13C in order to reconstruct past methane fluxes based on the lake sediment record.

  8. Azide and trans-cyclooctene dUTPs: incorporation into DNA probes and fluorescent click-labelling.

    PubMed

    Ren, Xiaomei; El-Sagheer, Afaf H; Brown, Tom

    2015-04-21

    5-Azidomethyl dUTP and two 5-trans-cyclooctene dUTPs with different linkers between the TCO and the uracil base have been incorporated into DNA by primer extension, reverse-transcription and PCR amplification. For azidomethyl dUTP the PCR reaction was successful even when the modified dUTP was not supplemented with dTTP. In one case 335 azidomethyl dU residues were incorporated into the 523 base pair amplicon using this methodology. 5-Azidomethyl dUTP was found to be a better substrate for DNA polymerases than the trans-cyclooctene dUTPs. However, the inverse electron demand Diels-Alder reaction between cyclooctene DNA and a tetrazine Cy3-dye was more efficient than the strain-promoted reaction between azide DNA and a bicyclo [6.1.0] non-4-yne Cy3 dye.

  9. Aliphatic (1)H, (13)C and (15)N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP(+) and folate.

    PubMed

    Loveridge, E Joel; Matthews, Stella M; Williams, Christopher; Whittaker, Sara B-M; Günther, Ulrich L; Evans, Rhiannon M; Dawson, William M; Crump, Matthew P; Allemann, Rudolf K

    2013-04-01

    Dihydrofolate reductase from the deep-sea bacterium Moritella profunda (MpDHFR) has been (13)C/(15)N isotopically labelled and purified. Here, we report the aliphatic (1)H, (13)C and (15)N resonance assignments of MpDHFR in complex with NADP(+) and folate. The spectra of MpDHFR suggest considerably greater conformational heterogeneity than is seen in the closely related DHFR from Escherichia coli.

  10. 13CO2 pulse labelling of plants in tandem with stable isotope probing: methodological considerations for examining microbial function in the rhizosphere.

    PubMed

    Griffiths, Robert I; Manefield, Mike; Ostle, Nick; McNamara, Niall; O'Donnell, Anthony G; Bailey, Mark J; Whiteley, Andrew S

    2004-07-01

    Recently developed 13CO2 pulse labelling and stable isotope probing (SIP) methods offer the potential to track 13C-labelled plant photosynthate into phylogenetic groups of microbial taxa in the rhizosphere, permitting an examination of the link between soil microbial diversity and carbon flow in situ. We tested the feasibility of this approach to detect functional differences in microbial communities utilising recently fixed plant photosynthate in moisture perturbed grassland turfs. Specifically, we addressed two questions: (1) How does moisture perturbation (three treatments; continual wetting, drying, and drying followed by rewetting) affect the assimilation of 13C-labelled exudates carbon into the soil microbial community?; (2) Can 13C deposited in soil from pulse-labelled plants be used to identify microbes utilising plant exudates using SIP methodologies? Net CO2 fluxes showed that prior to 13CO2 pulse labelling, all treatments were photosynthetically active, but differences were observed in night time respiration, indicating moisture treatments had impacted on net CO2 efflux. Measurements of pulse-derived 13C incorporated into soil RNA over 2 months showed that there was only evidence of 13C enrichment in the continuously wetted treatments. However, isotopic values represented only a 0.1-0.2 13C at.% increase over natural abundance levels and were found to be insufficient for the application of RNA-SIP. These findings reveal that in this experimental system, the microbial uptake of labelled carbon from plant exudates is low, and further optimisation of methodologies may be required for application of SIP to natural plant-soil systems where 13C tracer dilution is a consideration. PMID:15177910

  11. Application of 13C Nuclear Magnetic Resonance To Elucidate the Unexpected Biosynthesis of Erythritol by Leuconostoc oenos

    PubMed Central

    Veiga-Da-Cunha, Maria; Firme, Paula; Romão, M. Vitória San; Santos, Helena

    1992-01-01

    Natural-abundance 13C nuclear magnetic resonance (13C-NMR) revealed the production of erythritol and glycerol by nongrowing cells of Leuconostoc oenos metabolizing glucose. The ratio of erythritol to glycerol was strongly influenced by the aeration conditions of the medium. The elucidation of the metabolic pathway responsible for erythritol production was achieved by 13C-NMR and 1H-NMR spectroscopy using specifically 13C-labelled d-glucose. The 1H-NMR spectrum of the cell supernatant resulting from the metabolism of [2-13C]glucose showed that only 75% of the glucose supplied was metabolized heterofermentatively and that the remaining 25% was channelled to the production of erythritol. The synthesis of this polyol resulted from the reduction of the C-4 moiety of the intermediate fructose 6-phosphate. Oxygen has an inhibitory effect on the production of erythritol by L. oenos. Preaeration of a suspension of nongrowing cells of L. oenos resulted in 30% less erythritol and in 70% more glycerol formed during the anaerobic metabolism of glucose. The anaerobic production of erythritol from glucose was also found in growing cultures of L. oenos, although to a smaller extent. PMID:16348738

  12. 13C NMR spectra of pyridine chalcone analogs

    NASA Astrophysics Data System (ADS)

    Jovanović, B. Ž.; Mišić-Vuković, M.; Marinković, A. D.; Csanádi, J.

    1999-05-01

    13C NMR spectra of two series of pyridine chalcone analogs were determined in deuterated dimethylsulphoxide (DMSO-d 6). It was established that these compounds were in more stable E-configurations except for the 4-pyridalacetophenone which was in Z-configuration. On the basis of the Hammett correlations of 13C NMR chemical shifts of the ethylenic bond carbon atoms and the σ values for the pyridine "aza" groups, the polarization of ethylenic bonds were discussed. It was established that the opposite effect of the pyridine substituents at the electronic density distribution in pyridalacetophenone and cinnamoylpyridines depends on its direct bonding to the ethylenic carbon or the transmission electronic effects via the carbonyl group, respectively.

  13. Metabolic Imaging in the Anesthetized Rat Brain Using Hyperpolarized [1-13C] Pyruvate and [1-13C] Ethyl Pyruvate

    PubMed Central

    Hurd, Ralph E.; Yen, Yi-Fen; Mayer, Dirk; Chen, Albert; Wilson, David; Kohler, Susan; Bok, Robert; Vigneron, Daniel; Kurhanewicz, John; Tropp, James; Spielman, Daniel; Pfefferbaum, Adolf

    2010-01-01

    Formulation, polarization, and dissolution conditions were developed to obtain a stable hyperpolarized solution of [1-13C]-ethyl pyruvate. A maximum tolerated concentration and injection rate were determined, and 13C spectroscopic imaging was used to compare the uptake of hyperpolarized [1-13C]-ethyl pyruvate relative to hyperpolarized [1-13C]-pyruvate into anesthetized rat brain. Hyperpolarized [1-13C]-ethyl pyruvate and [1-13C]-pyruvate metabolic imaging in normal brain is demonstrated and quantified in this feasibility and range-finding study. PMID:20432284

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

    NASA Astrophysics Data System (ADS)

    Hu, Kaifeng; Vögeli, Beat; Pervushin, Konstantin

    2005-06-01

    We propose the use of 13C-detected 3D HCC-TOCSY experiments for assignment of 1H and 13C resonances in protonated and partially deuterated proteins. The experiments extend 2D C-13-start and C-13-observe TOCSY type experiments proposed earlier [J. Biomol. NMR 26 (2) (2003) 167]. Introduction of the third 1H dimension to 2D TOCSY: (i) reduces the peak overlap and (ii) increases the sensitivity per unit time, even for highly deuterated (>85%) protein samples, which makes this improved method an attractive tool for the side-chain H and C assignment of average sized proteins with natural isotope abundance as well as large partially deuterated proteins. The experiments are demonstrated with a 16 kDa 15N, 13C-labeled non-deuterated apo-CcmE and a 48 kDa uniformly 15N, 13C-labeled and fractionally (˜90%) deuterated dimeric sFkpA. It is predicted that this method should be suitable for the assignment of methyl 13C and 1H chemical shifts of methyl protonated, highly deuterated and 13C-labeled proteins with even higher molecular weight.

  15. Short-term d13C changes in cultivated soils from Mexico

    NASA Astrophysics Data System (ADS)

    Lounejeva, E.; Etchevers, J.; Morales Puente, P.; Cienfuegos Alvarado, E.; Sedov, S.; Solleiro, E.; Hidalgo, C.

    2007-05-01

    (generally less than -20%o). The common feature for the soils under the Traditional and Traditional improved treatments was a C3 enrichment of the superficial SOM component compared to the underlying layers as a consequence of the dominance of the cultivated C3 species. A similar but more accentuated negative shift is also observed in the SOM from the forest soil (non-cultivated soil d13C -25.2), so the interpretation is uncertain. In the Traditional Organic treatment a clear and perceptible increment of d13C in the SOM carbon signature was observed. This was attributed mainly to the fact that cows manure may contain a lot of C4 coming from feedstuff rich in corn grain that is provided to the animals during grass shortage periods . However, the maize crop introduced in the rotation during the 3rd year had no major effect on the tepetates carbon isotopic signature. The stable isotopic carbon data corresponding to a short period (4 years) of observation in uniformly managed soil ecosystems showed that d13C changed due to the quality of the residues (relative abundance of C3/C4 species) incorporated to the SOC, but this memory is susceptible to undergo changes in the short term and could be rapidly reversed as a consequence of crop management.

  16. Development and testing of hyperpolarized 13C MR calibrationless parallel imaging

    NASA Astrophysics Data System (ADS)

    Feng, Yesu; Gordon, Jeremy W.; Shin, Peter J.; von Morze, Cornelius; Lustig, Michael; Larson, Peder E. Z.; Ohliger, Michael A.; Carvajal, Lucas; Tropp, James; Pauly, John M.; Vigneron, Daniel B.

    2016-01-01

    A calibrationless parallel imaging technique developed previously for 1H MRI was modified and tested for hyperpolarized 13C MRI for applications requiring large FOV and high spatial resolution. The technique was demonstrated with both retrospective and prospective under-sampled data acquired in phantom and in vivo rat studies. A 2-fold acceleration was achieved using a 2D symmetric EPI readout equipped with random blips on the phase encode dimension. Reconstructed images showed excellent qualitative agreement with fully sampled data. Further acceleration can be achieved using acquisition schemes that incorporate multi-dimensional under-sampling.

  17. Catabolism of glucose and lactose in Bifidobacterium animalis subsp. lactis, studied by 13C Nuclear Magnetic Resonance.

    PubMed

    González-Rodríguez, Irene; Gaspar, Paula; Sánchez, Borja; Gueimonde, Miguel; Margolles, Abelardo; Neves, Ana Rute

    2013-12-01

    Bifidobacteria are widely used as probiotics in several commercial products; however, to date there is little knowledge about their carbohydrate metabolic pathways. In this work, we studied the metabolism of glucose and lactose in the widely used probiotic strain Bifidobacterium animalis subsp. lactis BB-12 by in vivo (13)C nuclear magnetic resonance (NMR) spectroscopy. The metabolism of [1-(13)C]glucose was characterized in cells grown in glucose as the sole carbon source. Moreover, the metabolism of lactose specifically labeled with (13)C on carbon 1 of the glucose or the galactose moiety was determined in suspensions of cells grown in lactose. These experiments allowed the quantification of some intermediate and end products of the metabolic pathways, as well as determination of the consumption rate of carbon sources. Additionally, the labeling patterns in metabolites derived from the metabolism of glucose specifically labeled with (13)C on carbon 1, 2, or 3 in cells grown in glucose or lactose specifically labeled in carbon 1 of the glucose moiety ([1-(13)Cglucose]lactose), lactose specifically labeled in carbon 1 of the galactose moiety ([1-(13)Cgalactose]lactose), and [1-(13)C]glucose in lactose-grown cells were determined in cell extracts by (13)C NMR. The NMR analysis showed that the recovery of carbon was fully compatible with the fructose 6-phosphate, or bifid, shunt. The activity of lactate dehydrogenase, acetate kinase, fructose 6-phosphate phosphoketolase, and pyruvate formate lyase differed significantly between glucose and lactose cultures. The transcriptional analysis of several putative glucose and lactose transporters showed a significant induction of Balat_0475 in the presence of lactose, suggesting a role for this protein as a lactose permease. This report provides the first in vivo experimental evidence of the metabolic flux distribution in the catabolic pathway of glucose and lactose in bifidobacteria and shows that the bifid shunt is the only

  18. Catabolism of Glucose and Lactose in Bifidobacterium animalis subsp. lactis, Studied by 13C Nuclear Magnetic Resonance

    PubMed Central

    González-Rodríguez, Irene; Gaspar, Paula; Sánchez, Borja; Gueimonde, Miguel; Neves, Ana Rute

    2013-01-01

    Bifidobacteria are widely used as probiotics in several commercial products; however, to date there is little knowledge about their carbohydrate metabolic pathways. In this work, we studied the metabolism of glucose and lactose in the widely used probiotic strain Bifidobacterium animalis subsp. lactis BB-12 by in vivo 13C nuclear magnetic resonance (NMR) spectroscopy. The metabolism of [1-13C]glucose was characterized in cells grown in glucose as the sole carbon source. Moreover, the metabolism of lactose specifically labeled with 13C on carbon 1 of the glucose or the galactose moiety was determined in suspensions of cells grown in lactose. These experiments allowed the quantification of some intermediate and end products of the metabolic pathways, as well as determination of the consumption rate of carbon sources. Additionally, the labeling patterns in metabolites derived from the metabolism of glucose specifically labeled with 13C on carbon 1, 2, or 3 in cells grown in glucose or lactose specifically labeled in carbon 1 of the glucose moiety ([1-13Cglucose]lactose), lactose specifically labeled in carbon 1 of the galactose moiety ([1-13Cgalactose]lactose), and [1-13C]glucose in lactose-grown cells were determined in cell extracts by 13C NMR. The NMR analysis showed that the recovery of carbon was fully compatible with the fructose 6-phosphate, or bifid, shunt. The activity of lactate dehydrogenase, acetate kinase, fructose 6-phosphate phosphoketolase, and pyruvate formate lyase differed significantly between glucose and lactose cultures. The transcriptional analysis of several putative glucose and lactose transporters showed a significant induction of Balat_0475 in the presence of lactose, suggesting a role for this protein as a lactose permease. This report provides the first in vivo experimental evidence of the metabolic flux distribution in the catabolic pathway of glucose and lactose in bifidobacteria and shows that the bifid shunt is the only pathway

  19. MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model

    PubMed Central

    Serrao, Eva M; Kettunen, Mikko I; Rodrigues, Tiago B; Dzien, Piotr; Wright, Alan J; Gopinathan, Aarthi; Gallagher, Ferdia A; Lewis, David Y; Frese, Kristopher K; Almeida, Jaime; Howat, William J; Tuveson, David A; Brindle, Kevin M

    2016-01-01

    Objectives Pancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select patients for surgery. We investigated here whether pancreatic preneoplasia could be detected prior to the development of invasive cancers in genetically engineered mouse models of PCa using metabolic imaging. Design The concentrations of alanine and lactate and the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were measured in extracts prepared from the pancreas of animals at different stages of disease progression; from pancreatitis, through tissue with predominantly low-grade and then high-grade pancreatic intraepithelial neoplasia and then tumour. 13C magnetic resonance spectroscopic imaging (13C-MRSI) was used to measure non-invasively changes in 13C labelling of alanine and lactate with disease progression, following injection of hyperpolarised [1-13C]pyruvate. Results Progressive decreases in the alanine/lactate concentration ratio and ALT/LDH activity ratio with disease progression were accompanied by a corresponding decrease in the [1-13C]alanine/[1-13C]lactate signal ratio observed in 13C-MRSI images of the pancreas. Conclusions Metabolic imaging with hyperpolarised [1-13C]pyruvate enables detection and monitoring of the progression of PCa precursor lesions. Translation of this MRI technique to the clinic has the potential to improve the management of patients at high risk of developing PCa. PMID:26347531

  20. Two Techniques for Estimating Deglacial Mean-Ocean δ13 C Change from the Same Set of 493 Benthic δ13C Records

    NASA Astrophysics Data System (ADS)

    Peterson, C. D.; Lisiecki, L. E.; Gebbie, G.

    2013-12-01

    The crux of carbon redistribution over the deglaciation centers on the ocean, where the isotopic signature of terrestrial carbon (δ13C terrestrial carbon = -25‰) is observed as a 0.3-0.7‰ shift in benthic foraminiferal δ13C. Deglacial mean-ocean δ13C estimates vary due to different subsets of benthic δ13C data and different methods of weighting the mean δ13C by volume. Here, we present a detailed 1-to-1 comparison of two methods of calculating mean δ13C change and uncertainty estimates using the same set of 493 benthic Cibicidoides spp. δ13C measurements for the LGM and Late Holocene. The first method divides the ocean into 8 regions, and uses simple line fits to describe the distribution of δ13C data for each timeslice over 0.5-5 km depth. With these line fits, we estimate the δ13C value at 100-meter intervals and weight those estimates by the regional volume at each depth slice. The mean-ocean δ13C is the sum of these volume-weighted regional δ13C estimates and the uncertainty of these mean-ocean δ13C estimates is computed using Monte Carlo simulations. The whole-ocean δ13C change is estimated using extrapolated surface- and deep-ocean δ13C estimates, and an assumed δ13C value for the Southern Ocean. This method yields an estimated LGM-to-Holocene change of 0.38×0.07‰ for 0.5-5km and 0.35×0.16‰ for the whole ocean (Peterson et al., 2013, submitted to Paleoceanography). The second method reconstructs glacial and modern δ13C by combining the same data compilation as above with a steady-state ocean circulation model (Gebbie, 2013, submitted to Paleoceanography). The result is a tracer distribution on a 4-by-4 degree horizontal resolution grid with 23 vertical levels, and an estimate of the distribution's uncertainty that accounts for the distinct modern and glacial water-mass geometries. From both methods, we compare the regional δ13C estimates (0.5-5 km), surface δ13C estimates (0-0.5 km), deep δ13C estimates (>5 km), Southern Ocean

  1. Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed.

    PubMed

    Raap, J; Hollander, J; Ovchinnikova, T V; Swischeva, N V; Skladnev, D; Kiihne, S

    2006-08-01

    Interactions between (15)N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in (13)C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C(alpha) signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the (31)P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The (13)C-peptide peaks were selectively detected in a (13)C-detected (1)H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The (13)C-MAOSS results thus independently confirms previous findings from (15)N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution (13)C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary

  2. Simultaneous investigation of cardiac pyruvate dehydrogenase flux, Krebs cycle metabolism and pH, using hyperpolarized [1,2-(13)C2]pyruvate in vivo.

    PubMed

    Chen, Albert P; Hurd, Ralph E; Schroeder, Marie A; Lau, Angus Z; Gu, Yi-ping; Lam, Wilfred W; Barry, Jennifer; Tropp, James; Cunningham, Charles H

    2012-02-01

    (13)C MR spectroscopy studies performed on hearts ex vivo and in vivo following perfusion of prepolarized [1-(13)C]pyruvate have shown that changes in pyruvate dehydrogenase (PDH) flux may be monitored non-invasively. However, to allow investigation of Krebs cycle metabolism, the (13)C label must be placed on the C2 position of pyruvate. Thus, the utilization of either C1 or C2 labeled prepolarized pyruvate as a tracer can only afford a partial view of cardiac pyruvate metabolism in health and disease. If the prepolarized pyruvate molecules were labeled at both C1 and C2 positions, then it would be possible to observe the downstream metabolites that were the results of both PDH flux ((13)CO(2) and H(13)CO(3)(-)) and Krebs cycle flux ([5-(13)C]glutamate) with a single dose of the agent. Cardiac pH could also be monitored in the same experiment, but adequate SNR of the (13)CO(2) resonance may be difficult to obtain in vivo. Using an interleaved selective RF pulse acquisition scheme to improve (13)CO(2) detection, the feasibility of using dual-labeled hyperpolarized [1,2-(13)C(2)]pyruvate as a substrate for dynamic cardiac metabolic MRS studies to allow simultaneous investigation of PDH flux, Krebs cycle flux and pH, was demonstrated in vivo.

  3. Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline

    NASA Astrophysics Data System (ADS)

    Park, Ki Soo; Oh, Seung Soo; Soh, H. Tom; Park, Hyun Gyu

    2014-08-01

    A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 μM. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to

  4. Altered 13C glucose metabolism in the cortico–striato–thalamo–cortical loop in the MK-801 rat model of schizophrenia

    PubMed Central

    Eyjolfsson, Elvar M; Nilsen, Linn Hege; Kondziella, Daniel; Brenner, Eiliv; Håberg, Asta; Sonnewald, Ursula

    2011-01-01

    Using a modified MK-801 (dizocilpine) N-methyl--aspartic acid (NMDA) receptor hypofunction model for schizophrenia, we analyzed glycolysis, as well as glutamatergic, GABAergic, and monoaminergic neurotransmitter synthesis and degradation. Rats received an injection of MK-801 daily for 6 days and on day 6, they also received an injection of [1-13C]glucose. Extracts of frontal cortex (FCX), parietal and temporal cortex (PTCX), thalamus, striatum, nucleus accumbens (NAc), and hippocampus were analyzed using 13C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, and gas chromatography–mass spectrometry. A pronounced reduction in glycolysis was found only in PTCX, in which 13C labeling of glucose, lactate, and alanine was decreased. 13C enrichment in lactate, however, was reduced in all areas investigated. The largest reductions in glutamate labeling were detected in FCX and PTCX, whereas in hippocampus, striatum, and Nac, 13C labeling of glutamate was only slightly but significantly reduced. The thalamus was the only region with unaffected glutamate labeling. γ-Aminobutyric acid (GABA) labeling was reduced in all areas, but most significantly in FCX. Glutamine and aspartate labeling was unchanged. Mitochondrial metabolites were also affected. Fumarate labeling was reduced in FCX and thalamus, whereas malate labeling was reduced in FCX, PTCX, striatum, and NAc. Dopamine turnover was decreased in FCX and thalamus, whereas that of serotonin was unchanged in all regions. In conclusion, neurotransmitter metabolism in the cortico–striato–thalamo–cortical loop is severely impaired in the MK-801 (dizocilpine) NMDA receptor hypofunction animal model for schizophrenia. PMID:21081956

  5. Pathway analysis using (13) C-glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila.

    PubMed

    Häuslein, Ina; Manske, Christian; Goebel, Werner; Eisenreich, Wolfgang; Hilbi, Hubert

    2016-04-01

    Amino acids represent the prime carbon and energy source for Legionella pneumophila, a facultative intracellular pathogen, which can cause a life-threatening pneumonia termed Legionnaires' disease. Genome, transcriptome and proteome studies indicate that L. pneumophila also utilizes carbon substrates other than amino acids. We show here that glycerol promotes intracellular replication of L. pneumophila in amoeba or macrophages (but not extracellular growth) dependent on glycerol-3-phosphate dehydrogenase, GlpD. An L. pneumophila mutant strain lacking glpD was outcompeted by wild-type bacteria upon co-infection of amoeba, indicating an important role of glycerol during infection. Isotopologue profiling studies using (13) C-lab