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Sample records for 13c labeled samples

  1. Synthesis of [13C6]-labelled phenethylamine derivatives for drug quantification in biological samples.

    PubMed

    Karlsen, Morten; Liu, HuiLing; Berg, Thomas; Johansen, Jon Eigill; Hoff, Bård Helge

    2014-05-15

    The availability of high-quality (13)C-labelled internal standards will improve accurate quantification of narcotics and drugs in biological samples. Thus, the synthesis of 10 [(13)C6]-labelled phenethylamine derivatives, namely amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 4-methoxyamphetamine, 4-methoxymethamphetamine, 3,5-dimethoxyphenethylamine 4-bromo-2,5-dimethoxyphenethylamine and 2,5-dimethoxy-4-iodophenethylamine, have been undertaken. [(13)C6]-Phenol proved to be an excellent starting material for making (13)C-labelled narcotic substances in the phenethylamine class, and a developed Stille-type coupling enabled an efficient synthesis of the 3,4-methylenedioxy and 4-methoxy derivatives. The pros and cons of alternative routes and transformations are also discussed. The [(13)C6]-labelled compounds are intended for use as internal standards in forensic analysis, health sciences and metabolomics studies by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. PMID:24634286

  2. 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

  3. 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

  4. IRMS detection of testosterone manipulated with 13C labeled standards in human urine by removing the labeled 13C.

    PubMed

    Wang, Jingzhu; Yang, Rui; Yang, Wenning; Liu, Xin; Xing, Yanyi; Xu, Youxuan

    2014-12-10

    Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ(13)C value). However, (13)C labeled standards can be used to control the δ(13)C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the (13)C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ(13)C values between Andro and ANAD (Δδ(13)CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different (13)C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ(13)CAndro-ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ(13)CAndro-ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-(13)C labeled standards. PMID:25441891

  5. Influence of 13C isotopic labeling location of 13C DNP of acetate using TEMPO free radical

    NASA Astrophysics Data System (ADS)

    Parish, Christopher; Niedbalski, Peter; Lumata, Lloyd

    2015-03-01

    Dynamic nuclear polarization (DNP) via the dissolution method enhances the liquid-state magnetic resonance (NMR or MRI) signals of insensitive nuclear spins by at least 10,000-fold. The basis for all these signal enhancements at room temperature is the polarization transfer from the electrons to nuclear spins at cryogenic temperature and high magnetic field. In this work, we have studied the influence of the location of 13C isotopic labeling on the DNP of sodium acetate at 3.35 T and 1.4 K using a wide ESR linewidth free radical 4-oxo-TEMPO. The carbonyl [1-13C]acetate spins produced a polarization level that is almost twice that of the methyl [2-13C]acetate spins. On the other hand, the polarization of the methyl 13C spins doubled to reach the level of [1-13C]acetate when the methyl group was deuterated. Meanwhile, the solid-state nuclear relaxation of these samples are the same and do not correlate with the polarization levels. These behavior implies that the nuclear relaxation for these samples is dominated by the contribution from the free radicals and the polarization levels can be explained by a thermodynamic picture of DNP.

  6. Strategy for Enhancement of (13)C-Photo-CIDNP NMR Spectra by Exploiting Fractional (13)C-Labeling of Tryptophan.

    PubMed

    Eisenreich, Wolfgang; Joshi, Monika; Illarionov, Boris; Kacprzak, Sylwia; Lukaschek, Michail; Kothe, Gerd; Budisa, Nediljko; Fischer, Markus; Bacher, Adelbert; Weber, Stefan

    2015-10-29

    The photo-CIDNP effect has proven to be useful to strongly enhance NMR signals of photochemically active proteins simply by irradiation with light. The evolving characteristic patterns of enhanced absorptive and emissive NMR lines can be exploited to elucidate the photochemistry and photophysics of light-driven protein reactions. In particular, by the assignment of (13)C NMR resonances, redox-active amino acids may be identified and thereby electron-transfer pathways unraveled, in favorable cases, even with (13)C at natural abundance. If signal enhancement is weak, uniform (13)C isotope labeling is traditionally applied to increase the signal strength of protein (13)C NMR. However, this typically leads to cross relaxation, which transfers light-induced nuclear-spin polarization to adjacent (13)C nuclei, thereby preventing an unambiguous analysis of the photo-CIDNP effect. In this contribution, two isotope labeling strategies are presented; one leads to specific but ubiquitous (13)C labeling in tryptophan, and the other is based on fractional isotope labeling affording sets of isotopologs with low probability of next-neighbor isotope accumulation within individual tryptophan molecules. Consequently, cross relaxation is largely avoided while the signal enhancement by (13)C enrichment is preserved. This results in significantly simplified polarization patterns that are easier to analyze with respect to the generation of light-generated nuclear-spin polarization. PMID:26244593

  7. Interlobe communication in 13C-methionine-labeled human transferrin.

    PubMed

    Beatty, E J; Cox, M C; Frenkiel, T A; Tam, B M; Mason, A B; MacGillivray, R T; Sadler, P J; Woodworth, R C

    1996-06-18

    [1H, 13C] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These are thought to play an important role in the recognition of this protein by its cellular receptors. [1H, 13C] NMR resonance assignments are presented for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [1H, 13C] HMQC spectra, and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine 13CH3 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subsequently to the N-lobe. The NMR shifts of Met464, which is in the Trp460-centered hydrophobic patch of helix 5 in the C-lobe in contact with the anion and metal binding site, show that Ga3+ binding causes movement of side chains within this helix, as is also the case in the N-lobe. The C-lobe residue Met382, which contacts the N-lobe hinge region, is perturbed when Ga3+ binds to the N-lobe, indicative of interlobe communication, a feature which may control the recognition of fully-metallated transferrin by its receptor. These results demonstrate that selective 13C labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins. PMID:8672464

  8. 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

    2016-04-20

    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. PMID:26778667

  9. Accurate determinations of one-bond 13C-13C couplings in 13C-labeled carbohydrates

    NASA Astrophysics Data System (ADS)

    Azurmendi, Hugo F.; Freedberg, Darón I.

    2013-03-01

    Carbon plays a central role in the molecular architecture of carbohydrates, yet the availability of accurate methods for 1DCC determination has not been sufficiently explored, despite the importance that such data could play in structural studies of oligo- and polysaccharides. Existing methods require fitting intensity ratios of cross- to diagonal-peaks as a function of the constant-time (CT) in CT-COSY experiments, while other methods utilize measurement of peak separation. The former strategies suffer from complications due to peak overlap, primarily in regions close to the diagonal, while the latter strategies are negatively impacted by the common occurrence of strong coupling in sugars, which requires a reliable assessment of their influence in the context of RDC determination. We detail a 13C-13C CT-COSY method that combines a variation in the CT processed with diagonal filtering to yield 1JCC and RDCs. The strategy, which relies solely on cross-peak intensity modulation, is inspired in the cross-peak nulling method used for JHH determinations, but adapted and extended to applications where, like in sugars, large one-bond 13C-13C couplings coexist with relatively small long-range couplings. Because diagonal peaks are not utilized, overlap problems are greatly alleviated. Thus, one-bond couplings can be determined from different cross-peaks as either active or passive coupling. This results in increased accuracy when more than one determination is available, and in more opportunities to measure a specific coupling in the presence of severe overlap. In addition, we evaluate the influence of strong couplings on the determination of RDCs by computer simulations. We show that individual scalar couplings are notably affected by the presence of strong couplings but, at least for the simple cases studied, the obtained RDC values for use in structural calculations were not, because the errors introduced by strong couplings for the isotropic and oriented phases are very

  10. 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

  11. 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

  12. A method to trace root-respired CO2 using a 13C label

    NASA Astrophysics Data System (ADS)

    Cooperdock, S.; Breecker, D.; Litvak, M. E.

    2014-12-01

    In order to partition total soil respiration into root respiration and decomposition under ambient conditions in desert soils, the following method was developed using 13C-labeled CO2 in a modern juniper savannah in central New Mexico. The labeled CO2 was mixed with ambient air and pumped into a small (2.5 m diameter and 1.4 m tall) juniper tree canopy . 10 L of the 13CO2 was sufficient to generate a stream of air at 20 L/min for 1 hour with a CO2 concentration of 540 ppm and a δ13C value of approximately 35,000‰. Plastic tarpaulins were used as a wind block. The 13CO2 -labeled air was applied to the canopy during peak photosynthesis between 10 and 11 am on June 30 2014 during which canopy air CO2 was elevated by approximately 10 ppm over ambient and had δ13C values ranging from 50 to 1000 ‰. Over the next three days, gas and tissue samples were collected in order to trace the 13C label through the juniper tree. Leaf and root samples collected from the labeled tree and from several control trees were loaded into exetainer vials, flushed with CO2-free air and incubated in the dark for 5 hours in order to measure the carbon isotope composition of respired CO2. Samples of soil pore space gas were collected from wells under the labeled tree and a control tree and were transported to the laboratory in He-flushed exetainer vials. The δ13C values of CO2 in the soil gas samples and in the headspace of incubation vials were measured using an isotope ratio mass spectrometer. The δ13C values of foliar respiration were significantly higher than those of the control (by 3.6‰, p < 0.01) one and two days after labeling and δ13C values of root-respired CO2 were significantly higher (by 0.7‰, p = 0.01) than those of the control three days after labeling. In addition, δ13C values of soil respired CO2, determined from measurements of soil pore space CO2 at 50 cm three days after labeling, were significantly higher (by 0.7‰, p < 0.03)) for the labeled tree than control

  13. Oxidation of 13C-labeled methane in surface crusts of pig- and cattle slurry.

    PubMed

    Ambus, Per; Petersen, Søren O

    2005-06-01

    Storage tanks for slurry from animal production constitute important point sources for emission of CH4 into the atmosphere. Recent investigations have demonstrated that surface crust formed on top of animal slurry provides a habitat for CH4 oxidation activity, a finding which may open for new opportunities to reduce greenhouse gas emissions during storage of animal wastes. In this work, 13C-labeled CH4 was used as a tracer to examine the absolute rates of CH4 oxidation and production in intact crust materials, collected from six different pig- and cattle slurry tanks in late autumn. Methane concentrations were generally reduced in the presence of surface crust samples, with the exception of a LECA-based (light expanded clay aggregates) crust from a pig slurry tank. In four samples, CH4 consumption was induced following a 2-4 days lag phase, whereas one cattle slurry crust consumed CH4 immediately and showed a 92% decline in CH4 concentration within the first week. Consumption of 13C-labeled CH4 was paralleled by the production of 13C-labeled CO2, thus providing direct evidence that microbial oxidation of CH4 to CO2 was taking place. Between 23% and 36% of the CH4-13C consumed in the active samples was accounted for in the gas phase CO2 indicating incomplete conversion of CH4 to CO2; however, comparable amounts of 13C was immobilized in the crust samples. Overall, the results showed that significant CH4 oxidation to CO2 in slurry crust samples occurs immediately or is inducible upon exposure to CH4. PMID:16191764

  14. 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.

  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. 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. Biosynthetic production of universally (13)C-labelled polyunsaturated fatty acids as reference materials for natural health product research.

    PubMed

    Le, Phuong Mai; Fraser, Catherine; Gardner, Graeme; Liang, Wei-Wan; Kralovec, Jaroslav A; Cunnane, Stephen C; Windust, Anthony J

    2007-09-01

    Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope (13)C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, (13)C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, (13)C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-(13)C-labelled fatty acids using U-(13)C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of (13)C-labelled DHA has been achieved with high chemical purity ( >99%) and high (13)C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-(13)C-DHA was enzymatically re-esterified to

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

    DOEpatents

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

    2001-01-01

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

  19. 13c Measurements On Air of Small Ice Samples

    NASA Astrophysics Data System (ADS)

    Eyer, M.; Leuenberger, M.

    We have developed a new method for 13C analysis for very small air amounts of less than 0.5 cc STP, corresponding to less than 10 gram of ice. It is based on the needle-crasher technique, which we routinely use for CO2 concentration measurements by infrared laser absorption. The extracted air is slowly expanded into a large volume through a water trap held at ­100°C. This sampled air is then carried by a high helium flux through a modified Precon system of Thermo-Finnigan to separate CO2 from the air and to inject the pure CO2 gas in a low helium stream via an open split device to a Delta Plus XL mass spectrometer. The overall precision based on replicates of standard air is significantly better than 0.1 for a single analysis and is further improved by a triplicate measurement of the same sample through a specially designed gas splitter. We have used this new method for investigations on polar ice cores. The 13C measurements are important for climate reconstructions, e.g. to reconstruct the evolution and its variability in the terrestrial and oceanic carbon sinks and to identify natural variations in the marine carbon cycle. During the industrialization atmospheric 13C decreased by about -2, mainly due to the anthropogenic release of biogenic CO2 by fossil fuel burning. Reconstructions of carbon and oxygen cycles of Joos at al. [1999] using a double deconvolution method show that between 1930 and 1950 the net terrestrial release is changing to a net terrestrial uptake of CO2. A highly resolved 13C dataset of this time window would replenish the documentation of this behaviour. Further, it would be interesting to compare such data with O2/N2 measurements, known as an other partitioning tool for carbon sources and sinks. At the EGS 2002 we will present a highly resolved 13C record from Antarctic ice covering this time period.

  20. Predicting outcomes of steady-state 13C isotope tracing experiments using Monte Carlo sampling

    PubMed Central

    2012-01-01

    Background Carbon-13 (13C) analysis is a commonly used method for estimating reaction rates in biochemical networks. The choice of carbon labeling pattern is an important consideration when designing these experiments. We present a novel Monte Carlo algorithm for finding the optimal substrate input label for a particular experimental objective (flux or flux ratio). Unlike previous work, this method does not require assumption of the flux distribution beforehand. Results Using a large E. coli isotopomer model, different commercially available substrate labeling patterns were tested computationally for their ability to determine reaction fluxes. The choice of optimal labeled substrate was found to be dependent upon the desired experimental objective. Many commercially available labels are predicted to be outperformed by complex labeling patterns. Based on Monte Carlo Sampling, the dimensionality of experimental data was found to be considerably less than anticipated, suggesting that effectiveness of 13C experiments for determining reaction fluxes across a large-scale metabolic network is less than previously believed. Conclusions While 13C analysis is a useful tool in systems biology, high redundancy in measurements limits the information that can be obtained from each experiment. It is however possible to compute potential limitations before an experiment is run and predict whether, and to what degree, the rate of each reaction can be resolved. PMID:22289253

  1. Comprehensive signal assignment of 13C-labeled lignocellulose using multidimensional solution NMR and 13C chemical shift comparison with solid-state NMR.

    PubMed

    Komatsu, Takanori; Kikuchi, Jun

    2013-09-17

    A multidimensional solution NMR method has been developed using various pulse programs including HCCH-COSY and (13)C-HSQC-NOESY for the structural characterization of commercially available (13)C labeled lignocellulose from potatoes (Solanum tuberosum L.), chicory (Cichorium intybus), and corn (Zea mays). This new method allowed for 119 of the signals in the (13)C-HSQC spectrum of lignocelluloses to be assigned and was successfully used to characterize the structures of lignocellulose samples from three plants in terms of their xylan and xyloglucan structures, which are the major hemicelluloses in angiosperm. Furthermore, this new method provided greater insight into fine structures of lignin by providing a high resolution to the aromatic signals of the β-aryl ether and resinol moieties, as well as the diastereomeric signals of the β-aryl ether. Finally, the (13)C chemical shifts assigned in this study were compared with those from solid-state NMR and indicated the presence of heterogeneous dynamics in the polysaccharides where rigid cellulose and mobile hemicelluloses moieties existed together. PMID:24010724

  2. 13C-labelled microdialysis studies of cerebral metabolism in TBI patients☆

    PubMed Central

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

    2014-01-01

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

  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. 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

  5. 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

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

    PubMed Central

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

    1998-01-01

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

  7. 13C-NMR study of labeled vinyl groups in paramagnetic myoglobin derivatives.

    PubMed

    Sankar, S S; La Mar, G N; Smith, K M; Fujinari, E M

    1987-04-01

    The 13C-NMR spectra of high-spin met-aquo myoglobin, spin-equilibrium met-azido myoglobin, low-spin met-cyano myoglobin, deoxy myoglobin and carbonmonoxy myoglobin from sperm whale reconstituted with hemin 13C enriched at both vinyl alpha or beta positions have been recorded. In all cases the labeled vinyl 13C signals are clearly resolved and useful spectra could be obtained within approx. 15 minutes. The decoupling of multiplet structure due to attached proton(s) has led to the specific assignment of vinyl 13C alpha signals in all paramagnetic derivatives and the 13C beta signals in met-cyano myoglobin. In all other cases, the collapse of the proton multiplet structure as a function of 1H decoupling frequency has located, but not assigned, the attached 1H resonance positions which are obscured by the intense diamagnetic envelope in the 1H-NMR spectrum. The resulting vinyl 13C hyperfine shifts follow Curie behavior, and the patterns closely resemble those in the appropriate model complexes in the same oxidation/spin/ligation state, except that the protein exhibits more in-plane asymmetry. The hyperfine shift patterns are indicative of dominant pi contact shifts for all ferric complexes. Deoxy myoglobin vinyl 13C and 1H contact shifts provide little evidence for pi bonding. PMID:3828362

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

    PubMed

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

    1992-09-11

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

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

    PubMed

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

    2014-11-01

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

  10. HCCCH Experiment for Through-Bond Correlation of Thymine Resonances in 13C-Labeled DNA Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sklenář, Vladimír.; Masse, James E.; Feigon, Juli

    1999-04-01

    Application of heteronuclear magnetic resonance pulse methods to13C,15N-labeled nucleic acids is important for the accurate structure determination of larger RNA and DNA oligonucleotides and protein-nucleic acid complexes. These methods have been applied primarily to RNA, due to the availability of labeled samples. The two major differences between DNA and RNA are at the C2‧ of the ribose and deoxyribose and the additional methyl group on thymine versus uracil. We have enzymatically synthesized a13C,15N-labeled 32 base DNA oligonucleotide that folds to form an intramolecular triplex. We present two- and three-dimensional versions of a new HCCCH-TOCSY experiment that provides intraresidue correlation between the thymine H6 and methyl resonances via the intervening carbons (H6-C6-C5-Cme-Hme).

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

    PubMed Central

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

    2012-01-01

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

  12. 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%.

  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. Laboratory-scale production of 13C-labeled lycopene and phytoene by bioengineered Escherichia coli.

    PubMed

    Lu, Chi-Hua; Choi, Jin-Ho; Engelmann Moran, Nancy; Jin, Yong-Su; Erdman, John W

    2011-09-28

    Consumption of tomato products has been associated with decreased risks of chronic diseases such as cardiovascular disease and cancer, and therefore the biological functions of tomato carotenoids such as lycopene, phytoene, and phytofluene are being investigated. To study the absorption, distribution, metabolism, and excretion of these carotenoids, a bioengineered Escherichia coli model was evaluated for laboratory-scale production of stable isotope-labeled carotenoids. Carotenoid biosynthetic genes from Enterobacter agglomerans were introduced into the BL21Star(DE3) strain to yield lycopene. Over 96% of accumulated lycopene was in the all-trans form, and the molecules were highly enriched with 13C by 13C-glucose dosing. In addition, error-prone PCR was used to disrupt phytoene desaturase (crtI) function and create a phytoene-accumulating strain, which was also found to maintain the transcription of phytoene synthase (crtB). Phytoene molecules were also highly enriched with 13C when the 13C-glucose was the only carbon source. The development of this production model will provide carotenoid researchers a source of labeled tracer materials to further investigate the metabolism and biological functions of these carotenoids. PMID:21888370

  16. 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

  17. Metabolism of parenterally administered fat emulsions in the rat: studies of fatty acid oxidation with 1-13C- and 8-13C-labelled triolein.

    PubMed

    Bäurle, W; Brösicke, H; Matthews, D E; Pogan, K; Fürst, P

    1998-04-01

    To reassess the hypothesis that fatty acid catabolism occurs to completion via beta-oxidation, male Sprague-Dawley rats receiving continuous total parenteral nutrition (TPN) including 43% energy as fat were infused with [1-(13)C]- or [8-(13)C]triolein. Expired CO2 was collected continuously for 4 h and its 13C:12C ratio determined by isotope-ratio mass spectrometry. Bicarbonate retention was also assessed over 4 h by infusion of NaH14CO3 and measurement of the expired 14CO2. A possible loss of label from [8-(13)C]oleic acid from the citric acid cycle via labelled acetyl-CoA without oxidation to CO2 was assessed by infusing further animals with acetate labelled with 14C either at C atoms 1 or 2 and determination of its conversion to expired 14CO2. At isotopic steady state, 63.2 (SE 1.6)% (n 8) of the infused [1-(14)C]acetate and 46.0 (SE 1.2)% (n 8) of [2-(14)C]acetate was recovered as expired 14CO2. After correction for bicarbonate retention and non-oxidative isotope loss, 37.3 (SE 1.2)% (n 20) of the [1-(13)C]triolein was found to have been oxidized, whereas 32.6 (SE 1.0)% (n 20) of the [8-(13)C]triolein was oxidized (P < or = 0.01). The lower oxidation of the C atom at position 8 of oleic acid than that at position 1 indicates incomplete oxidative breakdown of the fatty acid after entering beta-oxidation. PMID:9624230

  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. 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

  20. 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.

  1. Respiration of 13C-Labeled Substrates Added to Soil in the Field and Subsequent 16S rRNA Gene Analysis of 13C-Labeled Soil DNA

    PubMed Central

    Padmanabhan, P.; Padmanabhan, S.; DeRito, C.; Gray, A.; Gannon, D.; Snape, J. R.; Tsai, C. S.; Park, W.; Jeon, C.; Madsen, E. L.

    2003-01-01

    Our goal was to develop a field soil biodegradation assay using 13C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived 13C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of 13C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for 13CO2 respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of 13CO2 emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF6, that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired 13CO2. Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of 13CO2 released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of 13C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with 13C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter

  2. 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

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

    SciTech Connect

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

    1991-09-15

    The edible alga Spirulina platensis was uniformly labeled with {sup 13}C by growth in an atmosphere of pure {sup 13}CO{sub 2}. 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 {sup 13}C-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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Effects of sampling method on foliar δ (13)C of Leymus chinensis at different scales.

    PubMed

    Liu, Yanjie; Li, Yan; Zhang, Lirong; Xu, Xingliang; Niu, Haishan

    2015-03-01

    Stable carbon isotope composition (δ (13)C) usually shows a negative relationship with precipitation at a large scale. We hypothesized that sampling method affects foliar δ (13)C and its response pattern to precipitation. We selected 11 sites along a precipitation gradient in Inner Mongolia and collected leaves of Leymus chinensis with five or six replications repeatedly in each site from 2009 to 2011. Additionally, we collected leaves of L. chinensis separately from two types of grassland (grazed and fenced) in 2011. Foliar δ (13)C values of all samples were measured. We compared the patterns that foliar δ (13)C to precipitation among different years or different sample sizes, the differences of foliar δ (13)C between grazed and fenced grassland. Whether actual annual precipitation (AAP) or mean annual precipitation (MAP), it was strongly correlated with foliar δ (13)C every year. Significant difference was found between the slopes of foliar δ (13)C to AAP and MAP every year, among the slopes of foliar δ (13)C to AAP from 2009 to 2011. The more samples used at each site the lower and convergent P-values of the linear regression test between foliar δ (13)C and precipitation. Furthermore, there was significant lower foliar δ (13)C value in presence of grazed type than fenced type grassland. These findings provide evidence that there is significant effect of sampling method to foliar δ (13)C and its response pattern to precipitation of L. chinensis. Our results have valuable implications in methodology for future field sampling studies. PMID:25798224

  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. In-Situ 13C-Labeling of Microbial Phospholipid Fatty Acids: Tracing Substrate Assimilation in a Petroleum-Contaminated Aquifer

    NASA Astrophysics Data System (ADS)

    Pombo, S. A.; Schroth, M. H.; Pelz, O.; Zeyer, J.

    2001-12-01

    Stable isotope analysis of phospholipid-derived fatty acids (PLFA) is a novel tool to trace assimilation of organic carbon in microbial communities. The 13C-labeling of biomarker fatty acids allows the identification of specific microbial populations involved in the metabolism of particular substrates, supplemented in 13C-labeled form. The goal of this study was to investigate the feasibility of 13C-labeling of PLFA and produced dissolved inorganic carbon (DIC) in a petroleum hydrocarbon (PHC)-contaminated aquifer during an in-situ experiment. To this end, we performed a single-well "push-pull" test in a monitoring well located in the denitrifying zone of a PHC-contaminated aquifer in Studen, Switzerland. During the experiment, we injected 500 L of site groundwater that was amended with 13C-labeled acetate (50% [2-13C]) and nitrate as reactants, and bromide as conservative tracer. Following the injection, we extracted a total of 1000 L of test solution/groundwater mixture after 4, 23 and 46 h from the same location. Concentrations of anions were measured in samples collected during the extraction. From these data, we computed first order rate coefficients for consumption of acetate (0.70 +/- 0.05 1/d) and nitrate (0.63 +/- 0.08 1/d). In addition, we extracted and identified PLFA, and measured \\delta13C values of PLFA and DIC. After only 4 h of incubation, we detected 13C-enrichment of certain PLFA in suspended biomass of extracted groundwater. After 46 h, we measured enrichments of up to 5000 per mil in certain PLFA (e.g. 16:1ω 7c), and up to 1500 per mil in the produced DIC. Our results demonstrate the feasibility of in-situ 13C-labeling of PLFA and DIC using push-pull tests to determine microbial activities in-situ in a natural ecosystem.

  11. Carbon transfer, partitioning and residence time in the plant-soil system: a comparison of two 13C-CO2 labelling techniques

    NASA Astrophysics Data System (ADS)

    Studer, Mirjam S.; Siegwolf, Rolf T. W.; Schmidt, Michael W. I.; Abiven, Samuel

    2014-05-01

    13C-CO2 labelling is a powerful tool to study the carbon (C) dynamics in plant-soil systems, whereby various approaches have been applied, differing in the duration of label exposure, the applied label strength and the sampling intervals. We made a direct comparison of the two main 13C-CO2 labelling techniques - pulse and continuous labelling - and evaluated if different approaches yield the same results regarding the C transfer time, C partitioning and the C residence time in different plant-soil compartments. We conducted a pulse labelling (exposure to 99 atom% 13C-CO2 for three hours, traced for eight days) and a continuous labelling (exposure to 10 atom% 13C-CO2, traced for 14 days) on identical plant-soil systems (Populus deltoides x nigra, Cambisol soil) and under controlled environmental conditions. The plant-soil systems were destructively harvested at five sampling dates, and the soil CO2 efflux was sampled throughout the experiments. The 13C distribution into leaves, petioles, stems, cuttings, roots, soil, microbial biomass and soil respiration was analysed and wee applied exponential (pulse labelling) and logistic (continuous labelling) functions to model the C dynamics. Our results confirm that pulse labelling is best suited to assess the minimum C transfer time, while continuous labelling can be applied to assess the C transfer through a compartment, including short-term storage pools. Both experiments yielded the same C partitioning patterns at the specific sampling days, however, the time of sampling was crucial. For example the results of belowground C partitioning were consistent only after eight days of labelling. The C mean residence times estimated by the rate constant of the exponential and logistic function were largely different for the two techniques, mostly due to the strong model assumptions (e.g. steady state). Pulse and continuous labelling techniques are both well suited to assess C cycling. With pulse labelling, the dynamics of fresh

  12. 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

  13. 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

  14. 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...

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

    SciTech Connect

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

    1982-01-01

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

  16. 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…

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

    PubMed

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

    2016-05-10

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

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

    PubMed

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

    2014-02-01

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

  19. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. 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

  3. 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.

  4. First airborne samples of a volcanic plume for δ13C of CO2 determinations

    NASA Astrophysics Data System (ADS)

    Fischer, Tobias P.; Lopez, Taryn M.

    2016-04-01

    Volcanic degassing is one of the main natural sources of CO2 to the atmosphere. Carbon isotopes of volcanic gases enable the determination of CO2 sources including mantle, organic or carbonate sediments, and atmosphere. Until recently, this work required sample collection from vents followed by laboratory analyses. Isotope ratio infrared analyzers now enable rapid analyses of plume δ13C-CO2, in situ and in real time. Here we report the first analyses of δ13C-CO2 from airborne samples. These data combined with plume samples from the vent area enable extrapolation to the volcanic source δ13C. We performed our experiment at the previously unsampled and remote Kanaga Volcano in the Western Aleutians. We find a δ13C source composition of -4.4‰, suggesting that CO2 from Kanaga is primarily sourced from the upper mantle with minimal contributions from subducted components. Our method is widely applicable to volcanoes where remote location or activity level precludes sampling using traditional methods.

  5. 13C measurements on organic aerosol - a comparison of sources with ambient samples

    NASA Astrophysics Data System (ADS)

    Dusek, Ulrike; Meusinger, Carl; Oyama, Beatriz; Holzinger, Rupert; Röckmann, Thomas

    2014-05-01

    The stable carbon isotopes 12C and 13C can be used to get information about sources and processing of organic aerosol (OA). We developed and tested a method to measure δ13C values of OA collected on filter samples in different volatility classes. These filter samples are introduced into an oven, where organic compounds are thermally desorbed in He at different temperatures. The compounds released at each temperature step are oxidized to CO2 using a platinum catalyst at 550 °C. The CO2 is then passed on to an isotope ratio mass spectrometer (IRMS) to measure d13C ratios. With a similar setup the chemical composition at each temperature step can be determined using a Proton Transfer Reaction Time-of-Flight Mass Spectrometer (PTR-ToF-MS). System evaluation with controlled test compounds showed that organic compounds usually start evaporating from the filter when their melting point is reached. Isotopic fractionation occurs only if one temperature step is within a few degrees of the melting point of the substance, so that the substance only partially evaporates. However, this effect should be limited in an ambient sample containing thousands of individual chemical compounds. δ13C values of aerosol filter samples do not depend on the sample amount used, i.e. the system shows good linearity. The reproducibility depends somewhat on the sample amount and is usually < ± 0.3 ‰ for oven temperatures up to 200 °C and < ± 0.5 ‰ for oven temperatures greater than 200 °C. We analysed aerosol samples collected in a tunnel in Brazil (vehicular emissions), laboratory generated secondary organic aerosol (SOA) from alpha-pinene ozonolysis, and ambient filter samples from a regional site in the Netherlands, an urban site in Belgium, and Sao Paulo Brazil. First results show that δ13C ratios of SOA and vehicular emissions do not change strongly with oven temperature, i.e. the more refractory organic compounds have similar isotopic composition as the more volatile compounds

  6. 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

  7. Exocrine pancreatic insufficiency: accuracy and clinical value of the uniformly labelled 13C-Hiolein breath test.

    PubMed Central

    Lembcke, B; Braden, B; Caspary, W F

    1996-01-01

    BACKGROUND AND AIMS: The 13C-Hiolein breath test (98% [U-13C] labelled long chain triglyceride mixture (highly labelled triolein) was evaluated as a non-invasive, non-radioactive test for exocrine pancreatic insufficiency. Accuracy and clinical validity were examined with reference to both the secretin pancreozymin test and faecal fat analysis. METHODS: A secretin pancreozymin test and faecal fat analysis were performed in 46 patients, 30 with exocrine pancreatic insufficiency and 16 with normal pancreatic function. In all of these patients and in seven healthy volunteers (controls), a 13C-Hiolein breath test was performed using 2 mg/kg [U-13C] labelled Hiolein with a standard risk snack (1.5 g/kg; 25% fat). 13CO2/12CO2 enrichment in the exhaled breath was measured by isotope ratio mass spectrometry. RESULTS: In patients with pancreatic steatorrhoea the 13CO2 response was below the 95% confidence interval of 13CO2 exhalation in the controls. These responses were also diminished (p < 0.001) compared with patients with impaired lipase output but normal fat excretion and with disease as well as healthy controls. There was a linear correlation between stimulated lipase output and the ratio of lipase output/13CO2 response (r = 0.95). Among the 40 patients in whom direct pancreatic function testing was clinically indicated, the sensitivity of the 13C-Hiolein test for detecting steatorrhoea was 91.7%, with a specificity of 85.7%. CONCLUSIONS: In patients with pancreatic disease the 13C-Hiolein breath test reflects impaired lipase output and indicates decompensated lipolysis. The 13C-Hiolein breath test is a convenient alternative to faecal fat analysis. PMID:9026480

  8. Microbial transformations of free versus sorbed alanine analyzed by position-specific 13C and 14C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Bore, Ezekiel; Kuzyakov, Yakov

    2015-04-01

    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 representative for the pool of LMWOS. To assess transformations of sorbed alanine, we combined position-specifically and uniformly 13C and 14C labeled alanine tracer solution with a loamy haplic luvisol that had previously been sterilized by γ-radiation. After shaking the mixtures, the supernatant was removed, as was all non-sorbed alanine by repeated shaking with millipore water. The labeled soil was added to non-sterilized soil from the same site. To compare the effect of sorption, soil labeled with the same position-specifically labeled tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time steps. The incorporation of 14C into microbial biomass was determined by CFE, and utilization of individual C positions by distinct microbial groups was evaluated by 13C-PLFA analysis. A dual peak in the respired CO2 revealed the influence of two sorption mechanisms. Microbial uptake and transformation of the sorbed alanine was 3 times slower compared to non-sorbed alanine. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we introduced the divergence index (DI). The DI reveals the convergent or divergent behaviour of C from individual molecule positions during microbial utilization. The DI revealed, that alanines C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFAs. This indicates

  9. Trimethylation Enhancement Using (13)C-Diazomethane ((13)C-TrEnDi): Increased Sensitivity and Selectivity of Phosphatidylethanolamine, Phosphatidylcholine, and Phosphatidylserine Lipids Derived from Complex Biological Samples.

    PubMed

    Canez, Carlos R; Shields, Samuel W J; Bugno, Magdalena; Wasslen, Karl V; Weinert, Hillary P; Willmore, William G; Manthorpe, Jeffrey M; Smith, Jeffrey C

    2016-07-19

    Significant sensitivity enhancements in the tandem mass spectrometry-based analysis of complex mixtures of several phospholipid classes has been achieved via (13)C-TrEnDi. (13)C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) lipids extracted from HeLa cells demonstrated greater sensitivity via precursor ion scans (PISs) than their unmodified counterparts. Sphingomyelin (SM) species exhibited neither an increased nor decreased sensitivity following modification. The use of isotopically labeled diazomethane enabled the distinction of modified PE and modified PC species that would yield isobaric species with unlabeled diazomethane. (13)C-TrEnDi created a PE-exclusive PIS of m/z 202.1, two PS-exclusive PISs of m/z 148.1 and m/z 261.1, and a PIS of m/z 199.1 for PC species (observed at odd m/z values) and SM species (observed at even m/z values). The standardized average area increase after TrEnDi modification was 10.72-fold for PE species, 2.36-fold for PC, and 1.05-fold for SM species. The sensitivity increase of PS species was not quantifiable, as there were no unmodified PS species identified prior to derivatization. (13)C-TrEnDi allowed for the identification of 4 PE and 7 PS species as well as the identification and quantitation of an additional 4 PE and 4 PS species that were below the limit of detection (LoD) prior to modification. (13)C-TrEnDi also pushed 24 PE and 6 PC lipids over the limit of quantitation (LoQ) that prior to modification were above the LoD only. PMID:27275841

  10. General last-step labeling of biomolecule-based substrates by [12C], [13C], and [11C] carbon monoxide.

    PubMed

    Cornilleau, Thomas; Audrain, Hélène; Guillemet, Aude; Hermange, Philippe; Fouquet, Eric

    2015-01-16

    Alkaloid-, steroid-, biotin-, carbohydrate-, nucleoside-, and peptide-based bioconjugates are easily labeled with CO by a last-step palladium-catalyzed carbonylation. The choice of the [(12)C], [(13)C], or [(11)C] isotope opens the way to a new class of potential tracers or ligands easily available for various applications. PMID:25562588

  11. [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

  12. 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

  13. NMR studies of bent DNA using {sup 13}C-enriched samples

    SciTech Connect

    Zimmer, D.P.; Crothers, D.M.

    1994-12-01

    Bending of the DNA double helix can be brought about by introducing runs of adenines (A-tracts) in phase with the helical repeat of the DNA. The requirements for bending of DNA by A-tracts are that the length of the A-tract be greater than 3 base pairs and that the A-tracts must be in phase with the helical repeat (every 10 or 11 bp). Other factors, such as the number of adenines in the run, flanking sequences, and whether the A-tracts are phased with respect to the 5{prime}A or the 3{prime}A, have effects upon the degree of bending as assayed by electrophoretic mobility on native polyacrylamide gels. There are a number of models for bending A-tract DNA. The junction-bending model postulates that the structure of A-tracts is similar to the fiber diffraction structure of poly A, in which there is a significant degree of base pair tilt with respect to the helix axis. In this model, bending occurs at the junction between the A-tract and the B-form helix to allow favorable stacking interactions to occur. The bend of the helix could arise as a result of some other perturbation of B-form DNA by A-tracts, such as propeller twist; bending also could be due to a combination of factors. Our goal is to find the structural features of A-tracts responsible for bending of the helix by performing NMR on oligonucleotides containing A-tracts to obtain higher resolution structural data. One of the problems encountered in NMR structure determination of nucleic acids and other macromolecules is the assignment of resonances to nuclei. This procedure can be greatly facilitated through the use of {sup 13}C-enriched nucleic acid samples. We are developing a technique for the enzymatic synthesis of labeled DNA for NMR. The technique we are developing is similar to RNA labeling techniques already in use. The technique involves growth of methylotrophic bacteria on {sup 13}CH{sub 3}OH.

  14. Comprehensive discovery of 13C labeled metabolites in the bacterium Methylobacterium extorquens AM1 using gas chromatography-mass spectrometry.

    PubMed

    Yang, Song; Hoggard, Jamin C; Lidstrom, Mary E; Synovec, Robert E

    2013-11-22

    Herein, we report the identification of isotopically labeled metabolite peaks (or the lack of labeling) between sets of GC-MS data from Methylobacterium extorquens AM1. M. extorquens AM1 is one of the best-characterized model organisms for the study of C1 metabolism in methylotrophic bacteria, a diverse group of microbes that can use reduced one-carbon (C1) sources, such as methanol and methane as a sole source for both energy generation and carbon assimilation. Application of a match value (MV) based metric was used to rank the metabolite peaks in the data from those exhibiting the most mass spectral indications of labeling, to those not exhibiting any indications of labeling. The MV-based ranking corresponded well with analyst interpretation of the mass spectra. The MV-based method was initially demonstrated and validated using a mixture of 21 standards with data sets generated for mixtures at natural abundance, a mixture with 6 of the compounds labeled, and a 1:1 mixture of the natural abundance and labeled mixtures. Experimental data from TMS-derivatized extracts from the bacterium M. extorquens AM1 grown with natural abundance or (13)C-labeled methanol as the carbon source were analyzed. Of 131 peaks considered for the analysis of M. extorquens AM1, the 40 peaks ranked highest for indications of (13)C labeling were all found to be labeled, while those peaks ranked lower progressed from peaks for which labeling was uncertain, to a larger number of peaks that were clearly not labeled. The list of peaks determined to be labeled forms a library of compounds that are known to be labeled following the methanol metabolic pathway in M. extorquens AM1 that can be further investigated in future work, e.g. fluxomic studies. PMID:24007683

  15. Nic1 Inactivation Enables Stable Isotope Labeling with 13C615N4-Arginine in Schizosaccharomyces pombe*

    PubMed Central

    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, 13C615N4-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 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable 13C615N4-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. PMID:25368411

  16. Evidence of the photosynthetic origin of monoterpenes emitted by quercus ilex L. leaves by {sup 13}C labeling

    SciTech Connect

    Loreto, F.; Ciccioli, P.; Cecinato, A.; Brancaleoni, E. |

    1996-04-01

    The carbon of the four main monoterpenes emitted by Quercus ilex L. leaves was completely labeled with {sup 13}C after a 20-min feeding with 99% {sup 13}CO{sub 2}. This labeling time course is comparable with the labeling time course of isoprene, the terpenoid emitted by other Quercus species and synthesized in leaf chloroplasts. It is also comparable with that of phosphoglyceric acid. Our experiment therefore provides evidence that monoterpenes emitted by Q. ilex are formed photosynthesis intermediates and may share the same synthetic pathway with isoprene. By analyzing the rate and the distribution of labeling in the different fragments, we looked for evidence of differential carbon labeling in the {alpha}-pinene emitted. However, the labeling pattern was quite uniform in the different fragments, suggesting that the carbon skeleton of the emitted monoterpenes comes from a unique carbon source. 16 refs., 3 figs., 1 tab.

  17. Analysis of defect structure in silicon. Characterization of samples from UCP ingot 5848-13C

    NASA Technical Reports Server (NTRS)

    Natesh, R.; Guyer, T.; Stringfellow, G. B.

    1982-01-01

    Statistically significant quantitative structural imperfection measurements were made on samples from ubiquitous crystalline process (UCP) Ingot 5848 - 13 C. Important trends were noticed between the measured data, cell efficiency, and diffusion length. Grain boundary substructure appears to have an important effect on the conversion efficiency of solar cells from Semix material. Quantitative microscopy measurements give statistically significant information compared to other microanalytical techniques. A surface preparation technique to obtain proper contrast of structural defects suitable for QTM analysis was perfected.

  18. 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

  19. 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

  20. Galacto-oligosaccharides have prebiotic activity in a dynamic in vitro colon model using a (13)C-labeling technique.

    PubMed

    Maathuis, Annet J H; van den Heuvel, Ellen G; Schoterman, Margriet H C; Venema, Koen

    2012-07-01

    Galacto-oligosaccharides (GOS) are considered to be prebiotic, although the contribution of specific members of the microbiota to GOS fermentation and the exact microbial metabolites that are produced upon GOS fermentation are largely unknown. We aimed to determine this using uniformly (13)C-labeled GOS. The normal (control) medium and unlabeled or (13)C-labeled GOS was added to a dynamic, validated, in vitro model of the large-intestine containing an adult-type microbiota. Liquid-chromatography MS was used to measure the incorporation of (13)C label into metabolites. 16S-rRNA stable isotope probing coupled to a phylogenetic micro-array was used to determine label incorporation in microbial biomass. The primary members within the complex microbiota that were directly involved in GOS fermentation were shown to be Bifidobacterium longum, B. bifidum, B. catenulatum, Lactobacillus gasseri, and L. salivarius, in line with the prebiotic effect of GOS, although some other species incorporated (13)C label also. GOS fermentation led to an increase in acetate (+49%) and lactate (+23%) compared with the control. Total organic acid production was 8.50 and 7.52 mmol/g of carbohydrate fed for the GOS and control experiments, respectively. At the same time, the cumulative production of putrefactive metabolites (branched-chain fatty acids and ammonia) was reduced by 55%. Cross-feeding of metabolites from primary GOS fermenters to other members of the microbiota was observed. Our findings support a prebiotic role for GOS and its potential to act as a synbiotic in combination with certain probiotic strains. PMID:22623395

  1. Carbon transfer from the host to Tuber melanosporum mycorrhizas and ascocarps followed using a 13C pulse-labeling technique.

    PubMed

    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 (13)CO2. The transfer of (13)C 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 (13)C and transferred it slowly to the mycorrhizas. The mycorrhizas first formed a carbon sink and accumulated (13)C prior to ascocarp development. Then, the mycorrhizas transferred (13)C to the ascocarps to provide constitutive carbon (1.7 mg of (13)C 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

  2. 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

  3. 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.

  4. 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

  5. Extreme 13C depletion of CCl2F2 in firn air samples from NEEM, Greenland

    NASA Astrophysics Data System (ADS)

    Zuiderweg, A.; Holzinger, R.; Martinerie, P.; Schneider, R.; Kaiser, J.; Witrant, E.; Etheridge, D.; Rubino, M.; Petrenko, V.; Blunier, T.; Röckmann, T.

    2012-07-01

    A series of 12 high volume air samples collected from the S2 firn core during the North Greenland Eemian Ice Drilling (NEEM) 2009 campaign have been measured for mixing ratio and stable carbon isotope composition of the chlorofluorocarbon CFC-12 (CCl2F2). While the mixing ratio measurements compare favorably to other firn air studies, the isotope results show extreme 13C depletion at the deepest measurable depth (65 m), to values lower than δ13C = -80‰ vs. VPDB (the international stable carbon isotope scale), compared to present day surface tropospheric measurements near -40‰. Firn air modeling was used to interpret these measurements. Reconstructed atmospheric time series indicate even larger depletions (to -120‰) near 1950 AD, with subsequent rapid enrichment of the atmospheric reservoir of the compound to the present day value. Mass-balance calculations show that this change must have been caused by a large change in the isotopic composition of anthropogenic CFC-12 emissions, probably due to technological changes in the CFC production process over the last 80 yr. Propagating the mass-balance calculations into the future demonstrates that as emissions decrease to zero, isotopic fractionation by the stratospheric sinks will lead to continued 13C enrichment in atmospheric CFC-12.

  6. 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

  7. 13C measurements on organic aerosol - ambient samples versus source studies

    NASA Astrophysics Data System (ADS)

    Dusek, Ulrike; Meusinger, Carl; Oyama, Beatriz; Ramon, Wichert; de Wilde, Peter A.; Holzinger, Rupert; Röckmann, Thomas

    2013-04-01

    The stable carbon isotopes 12C and 13C can be used to get information about sources and processing of organic aerosol (OA). We developed and tested a method to measure δ13C values of OA collected on filter samples in different volatility classes. These filter samples are introduced into an oven, where organic compounds are thermally desorbed in He at different temperatures. The compounds released at each temperature step are oxidized to CO2 using a platinum catalyst at 550 °C. The CO2 is then passed on to an isotope ratio mass spectrometer (IRMS) to measure δ13C ratios. With a similar setup the chemical composition at each temperature step can be determined using a Proton Transfer Reaction Time-of-Flight Mass Spectrometer (PTR-ToF-MS). System evaluation with controlled test compounds showed that organic compounds usually start evaporating from the filter when their melting point is reached. Isotopic fractionation occurs only, if one temperature step is within a few degrees of the melting point of the substance, so that the substance only partially evaporates. However, this effect should be limited in an ambient sample containing thousands of individual chemical compounds. We analysed aerosol samples collected in a tunnel in Brazil (vehicular emissions), laboratory generated secondary organic aerosol (SOA) from alpha-pinene ozonolysis, and ambient filter samples from a regional site in the Netherlands and an urban site in Belgium. First results indicate that SOA is more volatile than organic aerosol from ambient or tunnel filters. The δ13C ratios of SOA and vehicular emissions do not change strongly with oven temperature, i.e. the more refractory organic compounds have similar isotopic composition as the more volatile compounds. This is in contrast to ambient organic aerosol where the more volatile compounds evaporating below 200°C are depleted with respect to the refractory compounds. Possible reasons for this difference (mixture of sources vs the role of

  8. Recoupling of chemical shift anisotropies in solid-state NMR under high-speed magic-angle spinning and in uniformly 13C-labeled systems

    NASA Astrophysics Data System (ADS)

    Chan, Jerry C. C.; Tycko, Robert

    2003-05-01

    We demonstrate the possibility of recoupling chemical shift anisotropy (CSA) interactions in solid-state nuclear magnetic resonance (NMR) under high-speed magic-angle spinning (MAS) while retaining a static CSA powder pattern line shape and simultaneously attenuating homonuclear dipole-dipole interactions. CSA recoupling is accomplished by a rotation-synchronized radio-frequency pulse sequence with symmetry properties that permit static CSA line shapes to be obtained. We suggest a specific recoupling sequence, which we call ROCSA, for which the scaling factors for CSA and homonuclear dipole-dipole interactions are 0.272 and approximately 0.05, respectively. This sequence is suitable for high-speed 13C MAS NMR experiments on uniformly 13C-labeled organic compounds, including biopolymers. We demonstrate the ROCSA sequence experimentally by measuring the 13C CSA patterns of the uniformly labeled, polycrystalline compounds L-alanine and N-acetyl-D,L-valine at MAS frequencies of 11 and 20 kHz. We also present experimental data for amyloid fibrils formed by a 15-residue fragment of the β-amyloid peptide associated with Alzheimer's disease, in which four amino acid residues are uniformly labeled, demonstrating the applicability to biochemical systems of high molecular weight and significant complexity. Analysis of the CSA patterns in the amyloid fibril sample demonstrates the utility of ROCSA measurements as probes of peptide and protein conformation in noncrystalline solids.

  9. Soil microbial communities in a CO2-enriched and 13C-labelled treeline ecosystem with different tree species

    NASA Astrophysics Data System (ADS)

    Hiltbrunner, David; Hagedorn, Frank; Miltner, Anja; Schmidt, Michael W. I.

    2010-05-01

    The aim of this study was to estimate the responses of soil microbial communities at the alpine treeline to elevated CO2 and to gain insight into the C cycling through microbial groups under two tree species by tracking 13C signatures into phospholipid fatty acids (PLFA). In alpine treeline ecosystems, we exposed 30 year-old larch and pine trees growing on undisturbed thick mor-type organic layers to five years of elevated CO2 (+200 μmol CO2 mol-1) being depleted in 13C. Results showed that elevated CO2 increased soil respiration particularly under pine trees. However, we found negligible CO2 effects on the biomass and community structure of soil microorganisms, which might be due to small plant growth responses, and a comparatively small input of new plant-derived C into the thick organic layers with large C stocks. The tracing of 13C-depleted CO2 revealed that only a small portion of the microbial community actively metabolized new C (25%). The 13C label in individual PLFA indicated that mainly fungi were involved in the use of new substrate. Tree species affected soil microbial communities in the organic layer with a significantly higher ratio of fungal to bacterial fatty acids under pine than under larch trees. Under pine, fungal PLFA of the organic layer carried a stronger 13C label which strongly suggests a greater mycorrhizal activity that might also lead to the 60% greater input of new plant-derived C into soil organic matter under pine than under larch. In conclusion, our results show that significant responses of microbial communities in these treeline ecosystems if any would require more drastic and long lasting effects than five years of elevated CO2. Tree species have a major impact on the cycling of new plant C through soil microbial communities.

  10. 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

  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. 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.

  13. Non-targeted determination of (13)C-labeling in the Methylobacterium extorquens AM1 metabolome using the two-dimensional mass cluster method and principal component analysis.

    PubMed

    Reaser, Brooke C; Yang, Song; Fitz, Brian D; Parsons, Brendon A; Lidstrom, Mary E; Synovec, Robert E

    2016-02-01

    A novel analytical workflow is presented for the analysis of time-dependent (13)C-labeling of the metabolites in the methylotrophic bacterium Methylobacterium extorquens AM1 using gas chromatography time-of-flight mass spectrometry (GC-TOFMS). Using (13)C-methanol as the substrate in a time course experiment, the method provides an accurate determination of the number of carbons converted to the stable isotope. The method also extracts a quantitative isotopic dilution time course profile for (13)C uptake of each metabolite labeled that could in principle be used to obtain metabolic flux rates. The analytical challenges encountered require novel analytical platforms and chemometric techniques. GC-TOFMS offers advanced separation of mixtures, identification of individual components, and high data density for the application of advanced chemometrics. This workflow combines both novel and traditional chemometric techniques, including the recently reported two-dimensional mass cluster plot method (2D m/z cluster plot method) as well as principal component analysis (PCA). The 2D m/z cluster plot method effectively indexed all metabolites present in the sample and deconvoluted metabolites at ultra-low chromatographic resolution (RS≈0.04). Using the pure mass spectra extracted, two PCA models were created. Firstly, PCA was used on the first and last time points of the time course experiment to determine and quantify the extent of (13)C uptake. Secondly, PCA modeled the full time course in order to quantitatively extract the time course profile for each metabolite. The 2D m/z cluster plot method found 152 analytes (metabolites and reagent peaks), with 54 pure analytes, and 98 were convoluted, with 65 of the 98 requiring mathematical deconvolution. Of the 152 analytes surveyed, 83 were metabolites determined by the PCA model to have incorporated (13)C while 69 were determined to be either metabolites or reagent peaks that remained unlabeled. PMID:26787164

  14. The effect of biochar amendment on the soil microbial community - PLFA analyses and 13C labeling results

    NASA Astrophysics Data System (ADS)

    Watzinger, A.; Feichtmair, S.; Rempt, F.; Anders, E.; Wimmer, B.; Kitzler, B.; Zechmeister-Boltenstern, S.; Horacek, M.; Zehetner, F.; Kloss, S.; Richoz, S.; Soja, G.

    2012-04-01

    The effects of biochar amendment on plant growth and on the chemical / physical soil characteristics are well explored but only few studies have investigated the impact on soil microorganisms. The response of the soil microbial community to biochar amendment was investigated by phospholipid fatty acid (PLFA) analysis in (i) a large scale pot experiment, (ii) a small scale pot experiment using 13C labeled biochar and (iii) an incubation study using 13C labeled biochar. In the large scale pot experiment, three different agricultural soils from Austria (Planosol, Cambisol, Chernozem) and four different types of biochar were investigated. In total, 25 treatments with 5 replicates each were set up and monitored over a year. The results from the pot experiments showed no significant influence of biochar amendment on the total microbial biomass in the first 100 days after biochar addition. However, discriminant analysis showed a distinction of biochar and control soils as well as a strong effect of the pyrolysis temperature on the microbial composition. The effect of biochar was dependent on the type of soil. In the Planosol, some PLFAs were affected positively, especially when adding biochar with a low pyrolysis temperature, in the first month. In the long term, microbial community composition altered. Growth of fungi and gram negative bacteria was enhanced. In the Chernozem, PLFAs from various microbial groups decreased in the long term. Variability in the incubation study was low. Consequently, many PLFAs were significantly affected by biochar amendment. Again, in the Planosol, gram negative bacteria, actinomycetes and, after 2 weeks, gram positive bacteria increased under biochar amendment whereas in the chernozem total microbial biomass and gram positive bacteria were negatively affected in the long term. The 13C labeling studies confirmed the low degradability of the biochar, i.e. no alteration of the content and the δ13C in the soil organic matter within 100 days

  15. 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

  16. 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

  17. Long-term, Trans-Canada Decay of 13C-labelled Crop Residues

    NASA Astrophysics Data System (ADS)

    Ellert, B. H.; Janzen, H. H.; Gregorich, E. G.

    2009-05-01

    The balance between soil C inputs and outputs has important implications for agricultural sustainability and atmospheric composition. While considerable information is available on the short-term (2 to 20 months) decomposition of soil C inputs, the long-term decomposition and persistence remains a major gap in our understanding of carbon and nitrogen cycling in agroecosystems. In many biogeochemical models, assumptions about long-term decomposition are largely unverified. Many of the data available for long-term crop residue decomposition were collected before 1970 when radiocarbon-enriched materials were used. To address these gaps, we implemented a long-term, trans-Canada decay study to measure the decomposition (10 to 20 years) of barley (Hordeum vulgare) residues at ten sites across Canada's agricultural region. The barley residues were uniformly and highly enriched with the stable 13C isotope so that small amounts can be distinguished from background soil carbon. In this presentation we will discuss the rationale for the study, and explain how it was implemented and will be maintained. Because the study was initiated in the fall of 2007, we will present initial results on residue persistence during the early stages of crop residue decomposition. We will also discuss the potential for exploiting the 13C tracer to investigate the structural chemistry of stabilized soil organic matter, and the functional groups of organisms within the detrital community.

  18. Quantification of peptide m/z distributions from 13C-labeled cultures with high-resolution mass spectrometry.

    PubMed

    Allen, Doug K; Goldford, Joshua; Gierse, James K; Mandy, Dominic; Diepenbrock, Christine; Libourel, Igor G L

    2014-02-01

    Isotopic labeling studies of primary metabolism frequently utilize GC/MS to quantify (13)C in protein-hydrolyzed amino acids. During processing some amino acids are degraded, which reduces the size of the measurement set. The advent of high-resolution mass spectrometers provides a tool to assess molecular masses of peptides with great precision and accuracy and computationally infer information about labeling in amino acids. Amino acids that are isotopically labeled during metabolism result in labeled peptides that contain spatial and temporal information that is associated with the biosynthetic origin of the protein. The quantification of isotopic labeling in peptides can therefore provide an assessment of amino acid metabolism that is specific to subcellular, cellular, or temporal conditions. A high-resolution orbital trap was used to quantify isotope labeling in peptides that were obtained from unlabeled and isotopically labeled soybean embryos and Escherichia coli cultures. Standard deviations were determined by estimating the multinomial variance associated with each element of the m/z distribution. Using the estimated variance, quantification of the m/z distribution across multiple scans was achieved by a nonlinear fitting approach. Observed m/z distributions of uniformly labeled E. coli peptides indicated no significant differences between observed and simulated m/z distributions. Alternatively, amino acid m/z distributions obtained from GC/MS were convolved to simulate peptide m/z distributions but resulted in distinct profiles due to the production of protein prior to isotopic labeling. The results indicate that peptide mass isotopologue measurements faithfully represent mass distributions, are suitable for quantification of isotope-labeling-based studies, and provide additional information over existing methods. PMID:24387081

  19. Characterisation of black carbon-rich samples by (13)C solid-state nuclear magnetic resonance.

    PubMed

    Novotny, Etelvino H; Hayes, Michael H B; Deazevedo, Eduardo R; Bonagamba, Tito J

    2006-09-01

    There are difficulties in quantifying and characterising the organic matter (OM) in soils that contain significant amounts of partially oxidised char or charcoal materials. The anthropogenic black carbon (BC), such as that found in the Terra Preta de Indio soils of the Amazon region, is a good example of the OM that is difficult to analyse in such soils. (13)C direct polarisation/magic angle spinning (DP/MAS) at high MAS frequency, (1)H-(13)C cross polarisation (CP)/MAS with total suppression of spinning sidebands (TOSS), and chemical shift anisotropy (CSA) filter nuclear magnetic resonance techniques have been applied successfully for quantifying the different components of OM. However, because pyrogenic materials present strong local magnetic susceptibility heterogeneities, the use of CSA-filter and TOSS make the pulse sequences very sensitive to imperfections in the pi pulses. In this study, the DP/MAS pulse sequence was replaced by a CP with a radio frequency ramp--variable amplitude CP (VACP)--VACP/MAS pulse sequence, and composite pi pulses were used in the CSA-filter and TOSS pulse sequences. In that way, the component functionalities in a humic acid from a BC soil were successfully determined. The spectrometer time needed was greatly decreased by employing this VACP/MAS technique. This development provides an accurate method for characterising BC-rich samples from different origins. PMID:16688435

  20. Extreme 13C depletion of CCl2F2 in firn air samples from NEEM, Greenland

    NASA Astrophysics Data System (ADS)

    Zuiderweg, A.; Holzinger, R.; Martinerie, P.; Schneider, R.; Kaiser, J.; Witrant, E.; Etheridge, D.; Petrenko, V.; Blunier, T.; Röckmann, T.

    2013-01-01

    A series of 12 high volume air samples collected from the S2 firn core during the North Greenland Eemian Ice Drilling (NEEM) 2009 campaign have been measured for mixing ratio and stable carbon isotope composition of the chlorofluorocarbon CFC-12 (CCl2F2). While the mixing ratio measurements compare favorably to other firn air studies, the isotope results show extreme 13C depletion at the deepest measurable depth (65 m), to values lower than δ13C = -80‰ vs. VPDB (the international stable carbon isotope scale), compared to present day surface tropospheric measurements near -40‰. Firn air modeling was used to interpret these measurements. Reconstructed atmospheric time series indicate even larger depletions (to -120‰) near 1950 AD, with subsequent rapid enrichment of the atmospheric reservoir of the compound to the present day value. Mass-balance calculations show that this change is likely to have been caused by a large change in the isotopic composition of anthropogenic CFC-12 emissions, probably due to technological advances in the CFC production process over the last 80 yr, though direct evidence is lacking.

  1. Characterisation of black carbon-rich samples by 13C solid-state nuclear magnetic resonance

    NASA Astrophysics Data System (ADS)

    Novotny, Etelvino H.; Hayes, Michael H. B.; Deazevedo, Eduardo R.; Bonagamba, Tito J.

    2006-09-01

    There are difficulties in quantifying and characterising the organic matter (OM) in soils that contain significant amounts of partially oxidised char or charcoal materials. The anthropogenic black carbon (BC), such as that found in the Terra Preta de Índio soils of the Amazon region, is a good example of the OM that is difficult to analyse in such soils. 13C direct polarisation/magic angle spinning (DP/MAS) at high MAS frequency, 1H-13C cross polarisation (CP)/MAS with total suppression of spinning sidebands (TOSS), and chemical shift anisotropy (CSA) filter nuclear magnetic resonance techniques have been applied successfully for quantifying the different components of OM. However, because pyrogenic materials present strong local magnetic susceptibility heterogeneities, the use of CSA-filter and TOSS make the pulse sequences very sensitive to imperfections in the π pulses. In this study, the DP/MAS pulse sequence was replaced by a CP with a radio frequency ramp—variable amplitude CP (VACP)—VACP/MAS pulse sequence, and composite π pulses were used in the CSA-filter and TOSS pulse sequences. In that way, the component functionalities in a humic acid from a BC soil were successfully determined. The spectrometer time needed was greatly decreased by employing this VACP/MAS technique. This development provides an accurate method for characterising BC-rich samples from different origins.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

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

    2014-05-15

    The entitled monohydrolysis products, also known as α-ethylhexyl and β-ethylhexyl sulfosuccinate (EHSS), of the surfactant diisooctyl sulfosuccinate (DOSS) were synthesized in stable isotope-labelled form from [(13)C]4 -maleic anhydride. Sodium [(13)C]4 -1-carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate (α-EHSS) was prepared by the method of Larpent by reaction of 2-ethylhexan-1-ol with [(13)C]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 (13)C/(13)C (incredible natural abundance double quantum transfer) and (1)H/(13)C (heteronuclear multiple-bond correlation (HMBC)) NMR spectral correlation experiments. Sodium [(13)C]4 -2-carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate (β-EHSS) was prepared in four steps by reaction of 4-methoxybenzyl alcohol with [(13)C]4 -maleic anhydride, regioselective sodium bisulfite addition, N,N'-dicyclohexylcarbodiimide-mediated esterification with 2-ethylhexan-1-ol, and p-methoxybenzyl ester deprotection with trifluoroacetic acid (13% overall yield). The regiochemical outcome of the second synthesis was confirmed by a combination of (1)JCC scalar coupling constant analysis and (1)H/(13)C (HMBC) NMR spectral correlation. The materials prepared are required as internal standards for the liquid chromatography-mass spectrometry (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

  7. 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

  8. 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.

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

    PubMed

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

    1996-12-01

    HCN, a new 3D NMR technique for stepwise coherence transfer from 1H to 13C to 15N and reverse through direct spin couplings 1JCH and 1JCN, is presented as a method for detection and assignment of histidine and tryptophan side-chain 1H, 13C, and 15N resonances in uniformly 13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay tau 3 were employed for determination of optimal tau 3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD) at pH 6, H(C)N, the 1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-defined cross peaks in 20 h for the 13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 12 1H and 13C chemical shifts and 10 of the 12 15N chemical shifts were determined. The 13C dimension proved essential in assignment of the multiply overlapping 1H and 15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mM sample of phenylmethanesulfonyl fluoride (PMSF)-inhibited alpha-lytic protease (MW = 19.8 kD) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited alpha-lytic protease after 18 h at various temperatures ranging from 5 to 55 degrees C, probably due to efficient relaxation of active-site imidazole 1H and/or 15N nuclei. PMID:8995843

  10. {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.

  11. Microbial degradation of 13C-labeled 9-methylphenanthren in marine sediment

    SciTech Connect

    Nanny, M.A.; Bortiatynski, J.M.; Hatcher, P.G.; Selifonov, S.A.

    1996-12-31

    Microbial degradation of polycyclic aromatic hydrocarbons (PAHs) may serve as a natural means of mitigation for contaminated soils and sediments. In order for natural biodegradation to be a feasible remediation strategy, it is important to know the extent of degradation, the identity of degradation products, and their fate. It is also important to identify and characterize the portion of parent pollutant that becomes incorporated into the unextractable, insoluble fraction of soil or sediment. Does this fraction consist of the parent pollutant trapped within soil or sediments pores and in turn may be released slowly over time, or is it covalently bound with insoluble organic matter, or has it been converted into insoluble biomass? These are difficult questions to answer analytically, but they must be understood if microbial degradation is to be used as an effective remediation method. This paper presents results of biodegradation studies of carbon 13 labelled methyl phenanthrene by marine microbes in a contaminated marine sediment.

  12. Capillary Absorption Spectrometer for 13C Isotopic Composition of Pico to Subpico Molar Sample Quantities

    NASA Astrophysics Data System (ADS)

    Moran, J.; Kelly, J.; Sams, R.; Newburn, M.; Kreuzer, H.; Alexander, M.

    2011-12-01

    Quick incorporation of IR spectroscopy based isotope measurements into cutting edge research in biogeochemical cycling attests to the advantages of a spectroscopy versus mass spectrometry method for making some 13C measurements. The simple principles of optical spectroscopy allow field portability and provide a more robust general platform for isotope measurements. We present results with a new capillary absorption spectrometer (CAS) with the capability of reducing the sample size required for high precision isotopic measurements to the picomolar level and potentially the sub-picomolar level. This work was motivated by the minute sample size requirements for laser ablation isotopic studies of carbon cycling in microbial communities but has potential to be a valuable tool in other areas of biological and geological research. The CAS instrument utilizes a capillary waveguide as a sample chamber for interrogating CO2 via near IR laser absorption spectroscopy. The capillary's small volume (~ 0.5 mL) combined with propagation and interaction of the laser mode with the entire sample reduces sample size requirements to a fraction of that accessible with commercially available IR absorption including those with multi-pass or ring-down cavity systems. Using a continuous quantum cascade laser system to probe nearly adjacent rovibrational transitions of different isotopologues of CO2 near 2307 cm-1 permits sample measurement at low analyte pressures (as low as 2 Torr) for further sensitivity improvement. A novel method to reduce cw-fringing noise in the hollow waveguide is presented, which allows weak absorbance features to be studied at the few ppm level after averaging 1,000 scans in 10 seconds. Detection limits down to the 20 picomoles have been observed, a concentration of approximately 400 ppm at 2 Torr in the waveguide with precision and accuracy at or better than 1 %. Improvements in detection and signal averaging electronics and laser power and mode quality are

  13. 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.

  14. 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.

  15. 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.

  16. 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

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

    PubMed

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

    2016-09-01

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

  18. 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'. PMID:7533569

  19. 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

  20. Photochemically Induced Dynamic Nuclear Polarization Observed by Solid-State NMR in a Uniformly (13)C-Isotope-Labeled Photosynthetic Reaction Center.

    PubMed

    Paul, Shubhajit; Bode, Bela E; Matysik, Jörg; Alia, A

    2015-10-29

    A sample of solubilized and quinone-depleted reaction centers from the purple bacterium Rhodobacter (R.) sphaeroides wild type has been prepared entirely (13)C and (15)N isotope labeled at all positions of the protein as well as of the cofactors. In this sample, the occurrence of the solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect has been probed by (13)C solid-state magic-angle spinning NMR under illumination. Under continuous illumination, signal intensities are modified by the three-spin mixing (TSM) mechanism. Time-resolved illumination experiments reveal the occurrence of light-induced nuclear polarization on the time scale of hundreds of microseconds, initially dominated by the transient polarization of the singlet branch of the radical-pair mechanism. A first kinetic analysis shows that the lifetime of the polarization from the singlet branch, indicated by the enhanced absorptive intensities of the signals from aliphatic carbons, is significantly extended. Upon arrival of the polarization from the triplet decay branch, emissive polarization caused by the TSM mechanism is observed. Also, this arrival is significantly delayed. The decay of TSM polarization occurs in two steps, assigned to intra- and intermolecular spin diffusion. PMID:26110356

  1. 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.

  2. 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.

  3. Direct analysis of δ13C and concentration of dissolved organic carbon (DOC) in environmental samples by TOC-IRMS

    NASA Astrophysics Data System (ADS)

    Kirkels, Frédérique; Cerli, Chiara; Federherr, Eugen; Kalbitz, Karsten

    2014-05-01

    Dissolved organic carbon (DOC) plays an important role in carbon cycling in terrestrial and aquatic systems. Stable isotope analysis (delta 13C) of DOC could provide valuable insights in its origin, fluxes and environmental fate. Precise and routine analysis of delta 13C and DOC concentration are therefore highly desirable. A promising, new system has been developed for this purpose, linking a high-temperature combustion TOC analyzer trough an interface with a continuous flow isotope ratio mass spectrometer (Elementar group, Hanau, Germany). This TOC-IRMS system enables simultaneous stable isotope (bulk delta 13C) and concentration analysis of DOC, with high oxidation efficiency by high-temperature combustion for complex mixtures as natural DOC. To give delta 13C analysis by TOC-IRMS the necessary impulse for broad-scale application, we present a detailed evaluation of its analytical performance for realistic and challenging conditions inclusive low DOC concentrations and environmental samples. High precision (standard deviation, SD predominantly < 0.15 permil) and accuracy (R2 = 0.9997, i.e. comparison TOC-IRMS and conventional EA-IRMS) were achieved by TOC-IRMS for a broad diversity of DOC solutions. This precision is comparable or even slightly better than that typically reported for EA-IRMS systems, and improves previous techniques for δ13C analysis of DOC. Simultaneously, very good precision was obtained for DOC concentration measurements. Assessment of natural abundance and slightly 13C enriched DOC, a wide range of concentrations (0.2-150 mgC/L) and injection volumes (0.05-3 ml), demonstrated good analytical performance with negligible memory effects, no concentration/volume effects and a wide linearity. Low DOC concentrations (< 2 mgC/L), were correctly analyzed without any pre-concentration. Moreover, TOC-IRMS was successfully applied to analyze DOC from diverse terrestrial, freshwater and marine environments (SD < 0.23 permil). In summary, the TOC

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

    PubMed

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

    2015-10-01

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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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. PMID:26577022

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

    PubMed

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-07-01

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

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

    PubMed

    Thakur, Chandar S; Sama, Jacob N; Jackson, Melantha E; Chen, Bin; Dayie, T Kwaku

    2010-12-01

    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 (13)C-2-glycerol and (13)C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in (13)C-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 (13)C-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 (13)C-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 (13)C-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 CH(2)-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, which indicates

  13. Towards a vibrational analysis of spheroidene. Resonance Raman spectroscopy of 13C-labelled spheroidenes in petroleum ether and in the Rhodobacter sphaeroides reaction centre.

    PubMed

    Kok, P; Köhler, J; Groenen, E J; Gebhard, R; van der Hoef, I; Lugtenburg, J; Hoff, A F; Farhoosh, R; Frank, H A

    1994-04-28

    We report resonance Raman spectra of the carotenoid spheroidene and its 14'-13C and 15'-13C substituted analogues in petroleum ether and bound to the reaction centre of Rhodobacter sphaeroides R26. The spectra in petroleum ether correspond to planar all-trans spheroidene while those of the reaction centres are consistent with a nonplanar 15,15'-cis spheroidene. The effect of 13C labelling is largest in the carbon-carbon double-bond stretching region. The 15'-13C substitution of the reaction centre bound spheroidene, however, hardly changes the C=C band as compared to that for the natural abundance spheroidene apart from a new weak band at 1508 cm(-1). This observation has been interpreted as a decoupling of the C15=C15' stretch from the other double-bond stretches in combination with a small intrinsic Raman intensity of this local mode for 15,15'-cis spheroidene. PMID:8167135

  14. 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

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

    PubMed

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

    2012-06-01

    Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes. PMID:22543713

  16. The effect of sample hydration on 13C CPMAS NMR spectra of fulvic acids

    USGS Publications Warehouse

    Hatcher, P.G.; Wilson, M.A.

    1991-01-01

    Three fulvic acids, two of which have been well studied by a number of other groups (Armadale and Suwannee river fulvic acids) have been examined by high resolution solid-state 13C-NMR techniques to delineate the effect of absorbed water. Two main effects of absorbed water were observed: (1) changes in spin lattice relaxation times in the rotating frame and cross polarization times and (2) total loss of signal so that some fulvic acid is effectively in solution. These results suggest that discrepancies in the literature concerning observed relative signal intensities from different structural groups are due to absorbed water and emphasize the necessity for proper precautionary drying before spectroscopic analysis. ?? 1991.

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

    PubMed

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

    2016-05-01

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

  18. 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

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

    PubMed

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

    2015-06-01

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

  20. 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

  1. 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

  2. A capillary absorption spectrometer for stable carbon isotope ratio (13C/12C) analysis in very small samples

    NASA Astrophysics Data System (ADS)

    Kelly, J. F.; Sams, R. L.; Blake, T. A.; Newburn, M.; Moran, J.; Alexander, M. L.; Kreuzer, H.

    2012-02-01

    A capillary absorption spectrometer (CAS) suitable for IR laser isotope analysis of small CO2 samples is presented. The system employs a continuous-wave (cw) quantum cascade laser to study nearly adjacent rovibrational transitions of different isotopologues of CO2 near 2307 cm-1 (4.34 μm). This initial CAS system can achieve relative isotopic precision of about 10 ppm 13C, or ˜1‰ (per mil in delta notation relative to Vienna Pee Dee Belemnite) with 20-100 picomoles of entrained sample within the hollow waveguide for CO2 concentrations ˜400-750 ppm. Isotopic analyses of such gas fills in a 1-mm ID hollow waveguide of 0.8 m overall physical path length can be carried out down to ˜2 Torr. Overall 13C/12C ratios can be calibrated to ˜2‰ accuracy with diluted CO2 standards. A novel, low-cost method to reduce cw-fringing noise resulting from multipath distortions in the hollow waveguide is presented, which allows weak absorbance features to be studied at the few ppm level (peak-to-rms) after 1000 scans are co-added in ˜10 s. The CAS is meant to work directly with converted CO2 samples from a laser ablation-catalytic combustion micro-sampler to provide 13C/12C ratios of small biological isolates currently operating with spatial resolutions ˜50 μm.

  3. Organic vs. Conventional Grassland Management: Do 15N and 13C Isotopic Signatures of Hay and Soil Samples Differ?

    PubMed Central

    Klaus, Valentin H.; Hölzel, Norbert; Prati, Daniel; Schmitt, Barbara; Schöning, Ingo; Schrumpf, Marion; Fischer, Markus; Kleinebecker, Till

    2013-01-01

    Distinguishing organic and conventional products is a major issue of food security and authenticity. Previous studies successfully used stable isotopes to separate organic and conventional products, but up to now, this approach was not tested for organic grassland hay and soil. Moreover, isotopic abundances could be a powerful tool to elucidate differences in ecosystem functioning and driving mechanisms of element cycling in organic and conventional management systems. Here, we studied the δ15N and δ13C isotopic composition of soil and hay samples of 21 organic and 34 conventional grasslands in two German regions. We also used Δδ15N (δ15N plant - δ15N soil) to characterize nitrogen dynamics. In order to detect temporal trends, isotopic abundances in organic grasslands were related to the time since certification. Furthermore, discriminant analysis was used to test whether the respective management type can be deduced from observed isotopic abundances. Isotopic analyses revealed no significant differences in δ13C in hay and δ15N in both soil and hay between management types, but showed that δ13C abundances were significantly lower in soil of organic compared to conventional grasslands. Δδ15N values implied that management types did not substantially differ in nitrogen cycling. Only δ13C in soil and hay showed significant negative relationships with the time since certification. Thus, our result suggest that organic grasslands suffered less from drought stress compared to conventional grasslands most likely due to a benefit of higher plant species richness, as previously shown by manipulative biodiversity experiments. Finally, it was possible to correctly classify about two third of the samples according to their management using isotopic abundances in soil and hay. However, as more than half of the organic samples were incorrectly classified, we infer that more research is needed to improve this approach before it can be efficiently used in practice

  4. Human lactation: oxidation and maternal transfer of dietary (13)C-labelled α-linolenic acid into human milk.

    PubMed

    Demmelmair, Hans; Kuhn, Angelika; Dokoupil, Katharina; Hegele, Verena; Sauerwald, Thorsten; Koletzko, Berthold

    2016-06-01

    The origin of fatty acids in milk has not been elucidated in detail. We investigated the contribution of dietary α-linolenic acid (ALA) to human milk fat, its oxidation and endogenous conversion to long-chain polyunsaturated fatty acids. Ten lactating women were given (13)C-ALA orally, and breath and milk samples were collected for a five-day period, while dietary intakes were assessed. 37.5 ± 2.7 % (M ± SE) of the tracer was recovered in breath-CO2, and 7.3 ± 1.1 % was directly transferred into milk. About 0.25 % of the tracer was found in milk long-chain polyunsaturated fatty acids. Combining intake and milk data, we estimate that about 65 % of milk ALA is directly derived from maternal diet. Thus, the major portion of milk ALA is directly derived from the diet, but dietary ALA does not seem to contribute much as a precursor to milk n-3 long-chain polyunsaturated fatty acids within the studied time period. PMID:26444910

  5. Whey and casein labeled with L-[1-13C]leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion.

    PubMed

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon; Helmark, Ida C; Lund, Peter; Kristensen, Niels B; Frystyk, Jan; Flyvbjerg, Allan; Schjerling, Peter; van Hall, Gerrit; Kjaer, Michael; Holm, Lars

    2011-01-01

    Muscle protein turnover following resistance exercise and amino acid availability are relatively well described. By contrast, the beneficial effects of different sources of intact proteins in relation to exercise need further investigation. Our objective was to compare muscle anabolic responses to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after whey and casein intake, both of which were higher compared with control (P < 0.05). Phosphorylation of Akt and p70(S6K) was increased after exercise and protein intake (P < 0.05), but no differences were observed between the types of protein except for total 4E-BP1, which was higher after whey intake than after casein intake (P < 0.05). In conclusion, whey and casein intake immediately after resistance exercise results in an overall equal MPS response despite temporal differences in insulin and amino acid concentrations and 4E-BP1. PMID:21045172

  6. 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

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

    PubMed

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

    2012-02-01

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

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

    PubMed

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

    2011-02-01

    We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273-6279 (1982)), types of amino acids are labeled with (13)C or/and (15)N such that cross peaks between (13)CO(i - 1) and (15)NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with (13)C and the second with (15)N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B(2)R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin. PMID:21170670

  9. Identifying sources of methane sampled in the Arctic using δ13C in CH4 and Lagrangian particle dispersion modelling.

    NASA Astrophysics Data System (ADS)

    Cain, Michelle; France, James; Pyle, John; Warwick, Nicola; Fisher, Rebecca; Lowry, Dave; Allen, Grant; O'Shea, Sebastian; Illingworth, Samuel; Jones, Ben; Gallagher, Martin; Welpott, Axel; Muller, Jennifer; Bauguitte, Stephane; George, Charles; Hayman, Garry; Manning, Alistair; Myhre, Catherine Lund; Lanoisellé, Mathias; Nisbet, Euan

    2016-04-01

    An airmass of enhanced methane was sampled during a research flight at ~600 m to ~2000 m altitude between the North coast of Norway and Svalbard on 21 July 2012. The largest source of methane in the summertime Arctic is wetland emissions. Did this enhancement in methane come from wetland emissions? The airmass was identified through continuous methane measurements using a Los Gatos fast greenhouse gas analyser on board the UK's BAe-146 Atmospheric Research Aircraft (ARA) as part of the MAMM (Methane in the Arctic: Measurements and Modelling) campaign. A Lagrangian particle dispersion model (the UK Met Office's NAME model) was run backwards to identify potential methane source regions. This was combined with a methane emission inventory to create "pseudo observations" to compare with the aircraft observations. This modelling was used to constrain the δ13C CH4 wetland source signature (where δ13C CH4 is the ratio of 13C to 12C in methane), resulting in a most likely signature of -73‰ (±4‰7‰). The NAME back trajectories suggest a methane source region of north-western Russian wetlands, and -73‰ is consistent with in situ measurements of wetland methane at similar latitudes in Scandinavia. This analysis has allowed us to study emissions from remote regions for which we do not have in situ observations, giving us an extra tool in the determination of the isotopic source variation of global methane emissions.

  10. 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

  11. 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.

  12. 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

  13. Reductive carbonylation of aryl halides employing a two-chamber reactor: a protocol for the synthesis of aryl aldehydes including 13C- and D-isotope labeling.

    PubMed

    Korsager, Signe; Taaning, Rolf H; Lindhardt, Anders T; Skrydstrup, Troels

    2013-06-21

    A protocol has been developed for conducting the palladium-catalyzed reductive carbonylation of aryl iodides and bromides using 9-methylfluorene-9-carbonyl chloride (COgen) as a source of externally delivered carbon monoxide in a sealed two-chamber system (COware), and potassium formate as the in situ hydride source. The method is tolerant to a wide number of functional groups positioned on the aromatic ring, and it can be exploited for the isotope labeling of the aldehyde group. Hence, reductive carbonylations run with (13)COgen provide a facile access to (13)C-labeled aromatic aldehydes, whereas with DCO2K, the aldehyde is specifically labeled with deuterium. Two examples of double isotopic labeling are also demonstrated. Finally, the method was applied to the specific carbon-13 labeling of the β-amyloid binding compound, florbetaben. PMID:23692554

  14. A Capillary Absorption Spectrometer for Stable Carbon Isotope Ratio (13C/12C) Analysis in Very Small Samples

    SciTech Connect

    Kelly, James F.; Sams, Robert L.; Blake, Thomas A.; Newburn, Matthew K.; Moran, James J.; Alexander, M. L.; Kreuzer, Helen W.

    2012-02-06

    A capillary absorption spectrometer (CAS) suitable for IR laser isotope analysis of small CO{sub 2} samples is presented. The system employs a continuous-wave (cw) quantum cascade laser to study nearly adjacent rovibrational transitions of different isotopologues of CO{sub 2} near 2307 cm{sup -1} (4.34 {mu}m). This initial CAS system can achieve relative isotopic precision of about 10 ppm {sup 13}C, or {approx}1{per_thousand} (per mil in delta notation relative to Vienna Pee Dee Belemnite) with 20-100 picomoles of entrained sample within the hollow waveguide for CO{sub 2} concentrations {approx}400 to 750 ppm. Isotopic analyses of such gas fills in a 1-mm ID hollow waveguide of 0.8 m overall physical path length can be carried out down to {approx}2 Torr. Overall {sup 13}C/{sup 12}C ratios can be calibrated to {approx}2{per_thousand} accuracy with diluted CO{sub 2} standards. A novel, low-cost method to reduce cw-fringing noise resulting from multipath distortions in the hollow waveguide is presented, which allows weak absorbance features to be studied at the few ppm level (peak-to-rms) after 1,000 scans are co-added in {approx}10 sec. The CAS is meant to work directly with converted CO{sub 2} samples from a Laser Ablation-Catalytic-Combustion (LA CC) micro-sampler to provide {sup 13}C/{sup 12}C ratios of small biological isolates with spatial resolutions {approx}50 {mu}m.

  15. Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global 13C-labeled internal standards improve performance for quantitative metabolomics in bacteria

    PubMed Central

    Yang, Song; Sadilek, Martin; Lidstrom, Mary E.

    2010-01-01

    Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method in conjunction with accurate quantitation. To achieve this goal, a methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving a pentafluorophenylpropyl (PFPP) column and culture-derived global 13C-labeled internal standards (I.Ss.) has been developed and compared to hydrophilic interaction liquid chromatography (HILIC)-MS/MS and published combined two-dimensional gas chromatography and LC methods. All 50 tested metabolite standards from 5 classes (amino acids, carboxylic acids, nucleotides, acyl-CoAs and sugar phosphates) displayed good chromatographic separation and sensitivity on the PFPP column. In addition, many important critical pairs such as isomers / isobars (e.g. isoleucine / leucine, methylsuccinic acid / ethylmalonic acid and malonyl-CoA / 3-hydroxybutyryl-CoA) and metabolites of similar structure (e.g. malate / fumarate) were resolved better on the PFPP than on the HILIC column. Compared to only one 13C-labeled I.S., the addition of global 13C-labeled I.Ss. improved quantitative linearity and accuracy. PFPP-MS/MS with global 13C-labeled I.Ss. allowed the absolute quantitation of 42 metabolite pool sizes in M. extorquens AM1. A comparison of metabolite level changes published previously for ethylamine (C2) versus succinate (C4) cultures of Methylobacterium extorquens AM1 indicated a good consistency with the data obtained by PFPP-MS/MS, suggesting this single approach has the capability of providing comprehensive metabolite profiling similar to the combination of methods. The more accurate quantification obtained by this method forms a fundamental basis for flux measurements and can be used for metabolism modeling in bacteria in future studies. PMID:20950815

  16. 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

  17. Studies on the biodegradation of fosfomycin: synthesis of 13C-labeled intermediates, feeding experiments with Rhizobium huakuii PMY1, and isolation of labeled amino acids from cell mass by HPLC.

    PubMed

    McGrath, John W; Hammerschmidt, Friedrich; Kählig, Hanspeter; Wuggenig, Frank; Lamprecht, Günther; Quinn, John P

    2011-11-18

    Racemic (1R*,2R*)-1,2-dihydroxy-[1-(13)C(1)]propylphosphonic acid and 1-hydroxy-[1-(13)C(1)]acetone were synthesized and fed to R. huakuii PMY1. Alanine and a mixture of valine and methionine were isolated as their N-acetyl derivatives from the cell hydrolysate by reversed-phase HPLC and analyzed by NMR spectroscopy. It was found that the carbon atoms of the respective carboxyl groups were highly (13)C-labeled (up to 65 %). Hydroxyacetone is therefore considered an obligatory intermediate of the biodegradation of fosfomycin by R. huakuii PMY1. PMID:22012897

  18. Differentiating inflamed and normal lungs by the apparent reaction rate constants of lactate dehydrogenase probed by hyperpolarized 13C labeled pyruvate

    PubMed Central

    Xu, He N.; Kadlececk, Stephen; Shaghaghi, Hoora; Zhao, Huaqing; Profka, Harilla; Pourfathi, Mehrdad; Rizi, Rahim

    2016-01-01

    Background Clinically translatable hyperpolarized (HP) 13C-NMR can probe in vivo enzymatic reactions, e.g., lactate dehydrogenase (LDH)-catalyzed reaction by injecting HP 13C-pyruvate into the subject, which is converted to 13C labeled lactate by the enzyme. Parameters such as 13C-lactate signals and lactate-to-pyruvate signal ratio are commonly used for analyzing the HP 13C-NMR data. However, the biochemical/biological meaning of these parameters remains either unclear or dependent on experimental settings. It is preferable to quantify the reaction rate constants with a clearer physical meaning. Here we report the extraction of the kinetic parameters of the LDH reaction from HP 13C-NMR data and investigate if they can be potential predictors of lung inflammation. Methods Male Sprague-Dawley rats (12 controls, 14 treated) were used. One dose of bleomycin (2.5 U/kg) was administered intratracheally to the treatment group. The lungs were removed, perfused, and observed by the HP-NMR technique, where a HyperSense dynamic nuclear polarization system was used to generate the HP 13C-pyruvate for injecting into the lungs. A 20 mm 1H/13C dual-tuned coil in a 9.4-T Varian vertical bore NMR spectrometer was employed to acquire the 13C spectral data every 1 s over a time period of 300 s using a non-selective, 15-degree radiofrequency pulse. The apparent rate constants of the LDH reaction and their ratio were quantified by applying ratiometric fitting analysis to the time series data of 13C labeled pyruvate and lactate. Results The apparent forward rate constant kp=(3.67±3.31)×10−4 s−1, reverse rate constant kl=(4.95±2.90)×10−2 s−1, rate constant ratio kp/kl=(7.53±5.75)×10−3 for the control lungs; kp=(11.71±4.35)×10−4 s−1, kl=(9.89±3.89)×10−2 s−1, and kp/kl=(12.39±4.18)×10−3 for the inflamed lungs at the 7th day post treatment. Wilcoxon rank-sum test showed that the medians of these kinetic parameters of the 7-day cohort were significantly

  19. Use of 13C labeling to assess carbon partitioning in transgenic and nontransgenic (parental) rice and their rhizosphere soil microbial communities.

    PubMed

    Wu, Wei Xiang; Liu, Wei; Lu, Hao Hao; Chen, Ying Xu; Medha, Devare; Janice, Thies

    2009-01-01

    Photosynthetic assimilation of CO2 is a primary source of carbon in soil and root exudates and can influence the community dynamics of rhizosphere organisms. Thus, if carbon partitioning is affected in transgenic crops, rhizosphere microbial communities may also be affected. In this study, the temporal effects of gene transformation on carbon partitioning in rice and rhizosphere microbial communities were investigated under greenhouse conditions using the 13C pulse-chase labeling method and phospholipid fatty acid (PLFA) analysis. The 13C contents in leaves of transgenic (Bt) and nontransgenic (Ck) rice were significantly different at the seedling, booting and heading stages. There were no detectable differences in 13C distribution in rice roots and rhizosphere microorganisms at any point during rice development. Although a significantly lower amount of Gram-positive bacterial PLFAs and a higher amount of Gram-negative bacterial PLFAs were observed in Bt rice rhizosphere as compared with Ck at all plant development stages, there were no significant differences in the amount of individual 13C-PLFA between Bt and Ck rhizospheres at any growing stage. These findings indicate that the insertion of cry1Ab and marker genes into rice had no persistent or adverse effect on the photosynthate distribution in rice or the microbial community composition in its rhizosphere. PMID:19049503

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

    NASA Astrophysics Data System (ADS)

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

    2006-12-01

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

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

    PubMed

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

    2003-01-01

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

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

    PubMed

    Warren, J M; Iversen, C M; Garten, C T; Norby, R J; Childs, J; Brice, D; Evans, R M; Gu, L; Thornton, P; Weston, D J

    2012-06-01

    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 (13)CO(2) 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, sap flow, basal area growth, root growth and soil CO(2) efflux rate (CER) were assessed for each tree over a 3-week period. The progression of the (13)C label was concurrently tracked from the atmosphere through foliage, phloem, roots and surface soil CO(2) 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. Soil CER was strongly correlated with sap flow 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 HS treatment did not noticeably reduce the magnitude of belowground autotrophic and heterotrophic respiration based on surface soil CER, which was overwhelmingly driven by soil temperature and moisture. The (13)C label was immediately detected in foliage on label day (half-life = 0.5 day), progressed through phloem by Day 2 (half-life = 4.7 days), roots by Days 2-4, and subsequently was evident as respiratory release from soil which peaked between Days 3 and 6. The δ(13)C of soil CO(2) efflux was strongly correlated with phloem δ(13)C on the previous day, or 2 days earlier. While the (13)C label was readily tracked through the ecosystem, the fate of root C

  3. 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

  4. Carbon-proton scalar couplings in RNA. 3D heteronuclear and 2D isotope-edited NMR of a [sup 13]C-labeled extra-stable hairpin

    SciTech Connect

    Hines, J.V.; Landry, S.M.; Varani, G.; Tinoco, I. Jr. Lawrence Berkeley Lab., CA )

    1994-06-29

    Long range carbon-proton scalar couplings were measured for an RNA hairpin of 12 nucleotides using 3D and [sup 13]C-edited 2D NMR. The large one-bond carbon-proton scalar couplings ([sup 1]J[sub CH]) and small n-bond couplings ([sup 1]J[sub CH]) produce ECOSY type cross-peaks, thus facilitating the determination of the sign and magnitude of the smaller [sup 2]J[sub CH] or [sup 3]J[sub CH]. The UUCGRNA hairpin (5[prime]-rGGACUUCGGUCC-3[prime]), whose structure has been determined by our laboratory, was uniformly [sup 13]C-labeled at 30% isotopic enrichment. The observed [sup 1]J[sub CH] couplings were then correlated to the known structure. The signs of [sup 2]J[sub C4[prime]H5[prime

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

    PubMed

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

    2010-07-01

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

  6. 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.

  7. 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. PMID:25625825

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

    NASA Astrophysics Data System (ADS)

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

    1996-12-01

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

  9. 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

  10. 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

  11. 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.

  12. The use of δ13C isotope ratio mass spectrometry for methamphetamine profiling: comparison of ephedrine and pseudoephedrine-based samples to P2P-based samples.

    PubMed

    Toske, Steven G; Morello, David R; Berger, Jennifer M; Vazquez, Etienne R

    2014-01-01

    Differentiating methamphetamine samples produced from ephedrine and pseudoephedrine from phenyl-2-propanone precursors is critical for assigning synthetic route information for methamphetamine profiling. The use of isotope ratio mass spectrometry data is now a key component for tracking precursor information. Recent carbon (δ(13)C) isotope results from the analysis of numerous methamphetamine samples show clear differentiation for ephedrine and pseudoephedrine-produced samples compared to P2P-produced samples. The carbon isotope differences were confirmed from synthetic route precursor studies. PMID:24378294

  13. Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids.

    PubMed

    Wang, Hsin; He, Yan; Kroenke, Christopher D; Kodukula, Sarala; Storch, Judith; Palmer, Arthur G; Stark, Ruth E

    2002-04-30

    Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed. PMID:11969406

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The absorption and plasma elimination of vitamin K was investigated by uniformly labeling phylloquinone in kale with carbon-13 and 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 phylloquino...

  15. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  16. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  17. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  18. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  19. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  20. 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.

  1. Metabolomic profiling of 13C-labelled cellulose digestion in a lower termite: insights into gut symbiont function.

    PubMed

    Tokuda, Gaku; Tsuboi, Yuuri; Kihara, Kumiko; Saitou, Seikou; Moriya, Sigeharu; Lo, Nathan; Kikuchi, Jun

    2014-08-22

    Termites consume an estimated 3-7 billion tonnes of lignocellulose annually, a role in nature which is unique for a single order of invertebrates. Their food is digested with the help of microbial symbionts, a relationship that has been recognized for 200 years and actively researched for at least a century. Although DNA- and RNA-based approaches have greatly refined the details of the process and the identities of the participants, the allocation of roles in space and time remains unclear. To resolve this issue, a pioneer study is reported using metabolomics to chart the in situ catabolism of (13)C-cellulose fed to the dampwood species Hodotermopsis sjostedti. The results confirm that the secretion of endogenous cellulases by the host may be significant to the digestive process and indicate that a major contribution by hindgut bacteria is phosphorolysis of cellodextrins or cellobiose. This study provides evidence that essential amino acid acquisition by termites occurs following the lysis of microbial tissue obtained via proctodaeal trophallaxis. PMID:25009054

  2. 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

  3. Detection of adulteration in honey samples added various sugar syrups with 13C/12C isotope ratio analysis method.

    PubMed

    Tosun, Murat

    2013-06-01

    Honey can be adulterated in various ways. One of the adulteration methods is the addition of different sugar syrups during or after honey production. Starch-based sugar syrups, high fructose corn syrup (HFCS), glucose syrup (GS) and saccharose syrups (SS), which are produced from beet or canes, can be used for adulterating honey. In this study, adulterated honey samples were prepared with the addition of HFCS, GS and SS (beet sugar) at a ratio of 0%, 10%, 20%, 40% and 50% by weight. (13)C/(12)C analysis was conducted on these adulterated honey samples using an isotope ratio mass spectrometer in combination with an elemental analyser (EA-IRMS). As a result, adulteration using C(4) sugar syrups (HFCS and GS) could be detected to a certain extent while adulteration of honey using C(3) sugar syrups (beet sugar) could not be detected. Adulteration by using SS (beet sugar) still has a serious detection problem, especially in countries in which beet is used in manufacturing sugar. For this reason, practice and analysis methods are needed to meet this deficit and to detect the adulterations precisely in the studies that will be conducted. PMID:23411291

  4. 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...

  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. 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.

  7. 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

  8. 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

  9. Biogenic Volatile Organic Compound and Respiratory CO2 Emissions after 13C-Labeling: Online Tracing of C Translocation Dynamics in Poplar Plants

    PubMed Central

    Ghirardo, Andrea; Gutknecht, Jessica; Zimmer, Ina; Brüggemann, Nicolas; Schnitzler, Jörg-Peter

    2011-01-01

    Background Globally plants are the primary sink of atmospheric CO2, but are also the major contributor of a large spectrum of atmospheric reactive hydrocarbons such as terpenes (e.g. isoprene) and other biogenic volatile organic compounds (BVOC). The prediction of plant carbon (C) uptake and atmospheric oxidation capacity are crucial to define the trajectory and consequences of global environmental changes. To achieve this, the biosynthesis of BVOC and the dynamics of C allocation and translocation in both plants and ecosystems are important. Methodology We combined tunable diode laser absorption spectrometry (TDLAS) and proton transfer reaction mass spectrometry (PTR-MS) for studying isoprene biosynthesis and following C fluxes within grey poplar (Populus x canescens) saplings. This was achieved by feeding either 13CO2 to leaves or 13C-glucose to shoots via xylem uptake. The translocation of 13CO2 from the source to other plant parts could be traced by 13C-labeled isoprene and respiratory 13CO2 emission. Principal Finding In intact plants, assimilated 13CO2 was rapidly translocated via the phloem to the roots within 1 hour, with an average phloem transport velocity of 20.3±2.5 cm h−1. 13C label was stored in the roots and partially reallocated to the plants' apical part one day after labeling, particularly in the absence of photosynthesis. The daily C loss as BVOC ranged between 1.6% in mature leaves and 7.0% in young leaves. Non-isoprene BVOC accounted under light conditions for half of the BVOC C loss in young leaves and one-third in mature leaves. The C loss as isoprene originated mainly (76–78%) from recently fixed CO2, to a minor extent from xylem-transported sugars (7–11%) and from photosynthetic intermediates with slower turnover rates (8–11%). Conclusion We quantified the plants' C loss as respiratory CO2 and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux. PMID:21387007

  10. 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

  11. 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. PMID:14963630

  12. [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.

  13. 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

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

    PubMed Central

    2014-01-01

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

  15. Confirmation of K-Momentum Dark Exciton Vibronic Sidebands Using 13C-Labeled, Highly Enriched (6,5) Single-Walled Carbon Nanotubes

    SciTech Connect

    Blackburn, J. L.; Holt, J. M.; Irurzun, V. M.; Reasco, D. E.; Rumbles, G.

    2012-03-14

    A detailed knowledge of the manifold of both bright and dark excitons in single-walled carbon nanotubes (SWCNTs) is critical to understanding radiative and nonradiative recombination processes. Exciton-phonon coupling opens up additional absorption and emission channels, some of which may 'brighten' the sidebands of optically forbidden (dark) excitonic transitions in optical spectra. In this report, we compare {sup 12}C and {sup 13}C-labeled SWCNTs that are highly enriched in the (6,5) species to identify both absorptive and emissive vibronic transitions. We find two vibronic sidebands near the bright {sup 1}E{sub 11} singlet exciton, one absorptive sideband {approx}200 meV above, and one emissive sideband {approx}140 meV below, the bright singlet exciton. Both sidebands demonstrate a {approx}50 cm{sup -1} isotope-induced shift, which is commensurate with exciton-phonon coupling involving phonons of A'{sub 1} symmetry (D band, {omega} {approx} 1330 cm{sup -1}). Independent analysis of each sideband indicates that both sidebands arise from the same dark exciton level, which lies at an energy approximately 25 meV above the bright singlet exciton. Our observations support the recent prediction of, and mounting experimental evidence for, the dark K-momentum singlet exciton lying {approx}25 meV (for the (6,5) SWCNT) above the bright {Lambda}-momentum singlet. This study represents the first use of {sup 13}C-labeled SWCNTs highly enriched in a single nanotube species to unequivocally confirm these sidebands as vibronic sidebands of the dark K-momentum singlet exciton.

  16. 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. PMID:26803680

  17. Application of a nanoEA-IRMS system for δ13C measurement of biomineral-bound organics in samples of diatom opal with nanomolar quantities of C

    NASA Astrophysics Data System (ADS)

    Méndez-Vicente, Ana; María Mejía-Ramírez, Luz; Stoll, Heather

    2013-04-01

    We describe the isotopic measurement of δ13C in very small samples of diatom opal (nanomolar quantities of C) both from fossil sediments and cultures. We use a nano-EA system composed of a combustion elemental analyzer (EA3000 series, Eurovector), with standard 18 mm diameter quartz oxidation-reduction reactors and an ash removal device that aids in removal of uncombusted opal and ensures a long reactor lifetime. This is coupled to a custom designed trapping and cromatography system (Nano-CF, Nu Instruments Ltd.) which cryogenically removes CO2 generated by sample combustion and introduces the gas into a low-flow helium carrier stream to the mass spectrometer (Nu Perspective IRMS instrument, Nu Instruments Ltd.). This technique allows for an important reduction in the minimum sample requirements for analysis compared to a typical EA, however the need to reduce the contribution of the blank to the measured values becomes all the more critical. Blank from the capsules can be minimized through specific protocols including cleaning with solvents and reducing the size of the capsule by cutting it to a smaller size, attaining blanks as low as 13.75±2.15 nmol C. Under these conditions we can accurately measure both standards and diatom reference materials in the range of 100 to 330 nmol C, with a precision of 2σ < 1 ‰. The measured δ13C is independent of sample size in this range for standards or samples with δ13C < -11 ‰, which is the compositional range expected for natural diatom samples. Furthermore, no memory effect is observed in samples with an isotopic δ13C value differing by > 10 ‰ analysed in sequence. Applied to measure biomineral-bound organics in cleaned diatom samples from sediments, the low sample size requirements of this technique allows us to analyse multiple size fractions within one sample, and explore isotopic fractionation patterns between them. We have analysed samples from sediments of both centric and pennate diatoms typically in the

  18. Simultaneous determination of seven β2-agonists in human and bovine urine by isotope dilution liquid chromatography-tandem mass spectrometry using compound-specific minimally (13)C-labelled analogues.

    PubMed

    González-Antuña, Ana; Rodríguez-González, Pablo; Centineo, Giuseppe; García Alonso, J Ignacio

    2014-10-29

    Seven β2-agonist (clenproperol, clenbuterol, salbutamol, bronbuterol, ractopamine, clenpenterol and clencyclohexerol) were determined simultaneously in human and bovine urine by isotope dilution LC-ESI-MS/MS in a triple quadrupole instrument. The method is based on the application of multiple linear regression in combination with compound-specific minimally (13)C-labelled analogues. Additionally, the increase of the bandpass of the first quadrupole during the selected reaction monitoring (SRM) measurement procedure allowed the simultaneous quantification of the seven compounds at sub ngg(-1) levels in a single chromatogram without resorting to a methodological calibration graph. Recovery values at concentration levels between 5.0 and 0.05ngg(-1) ranged from 95 to 110% in fortified bovine urine and from 91 to 108% in human urine, with relative standard deviations lower than 5% except for salbutamol and ractopamine. The proposed methodology was validated by analyzing the certified reference material BCR-503 (lyophilized bovine urine) certified for clenbuterol and salbutamol. The limits of detection (LOD) for a sample volume of 10mL of both human and bovine urine was found to be lower than 0.012ngg(-1) for all compounds, except to salbutamol in bovine urine which was of 0.029ngg(-1). The use of compound-specific isotopically labelled analogues minimally labelled in (13)C minimized the occurrence of isotope effects and corrected for matrix effects during ESI ionization and can be efficiently applied for the quantification of ultra-trace concentrations of β2-agonists in human and bovine urine. PMID:25468499

  19. 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

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

    NASA Astrophysics Data System (ADS)

    Chugh, Jeetender; Hosur, Ramakrishna V.

    2008-10-01

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

  1. 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

  2. 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

    2015-01-01

    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 (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. PMID:26345303

  3. 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

  4. Atmospheric CO2 and its δ13C measurements from flask sampling at Lin'an regional background station in China

    NASA Astrophysics Data System (ADS)

    Xia, Lingjun; Zhou, Lingxi; Tans, Pieter P.; Liu, Lixin; Zhang, Gen; Wang, Hongyang; Luan, Tian

    2015-09-01

    This study presents CO2 concentrations of air flask samples obtained at a regional background station Lin'an (LAN) in China from January 2009 to December 2013, as well as their stable isotope ratios (δ13C) from January 2011 to December 2013. Background data is selected to represent background characteristics of atmospheric CO2 and δ13C over Yangzte River Delta. The annual mean background CO2 concentrations vary from 395.0 ppm in 2009 to 407.6 ppm in 2013, with a growth rate of 2.8 ± 0.1 ppm yr-1 over the study period, which is higher than the global average level. The annual mean values of δ13C vary from -8.55‰ in 2011 to -8.73‰ in 2013, with a growth rate of -0.029 ± 0.010‰ yr-1. Seasonal cycles of CO2 concentrations and δ13C are observed to be obvious at LAN. The minimum and maximum values of mean seasonal CO2 cycle appear in August and January, respectively, with the peak-to-peak amplitude of 19.4 ppm. The sign of δ13C seasonal cycle is opposite to that of CO2, and the peak-to-peak amplitude is 0.85‰. The isotopic signature of CO2 sources/sinks (δs) is also discussed in this paper. The estimated δs values for heating season (December-February) is -25.4‰ and for vegetative season is -21.3‰ (March-November) suggest the significant impact of fossil fuel combustions during winter heating season and biological activities during vegetative season.

  5. 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

  6. Stable isotope-enhanced two- and three-dimensional diffusion ordered 13C NMR spectroscopy (SIE-DOSY 13C NMR)

    NASA Astrophysics Data System (ADS)

    Vermillion, Karl; Price, Neil P. J.

    2009-06-01

    The feasibility of obtaining high quality homonuclear or heteronuclear diffusion-ordered 13C NMR data is shown to be greatly improved by using 13C isotopically-enriched samples. Stable isotope-enhanced diffusion ordered (SIE-DOSY) 13C NMR has been applied to 13C-enriched carbohydrates, and has been used to determine diffusion coefficients for pentose and hexose monosaccharides, and a disaccharide and trisaccharide. These 2D spectra were obtained with as little as 8 min of acquisition time. Fully resolved 3D DOSY-HMQC NMR spectra of [U- 13C]xylose, [U- 13C]glucose, and [1- 13C gal]lactose were obtained in 5 h. Sample derivatization with [ carbonyl- 13C]acetate (peracetylation) extends the usefulness of the technique to included non-labeled sugars; the 13C-carbonyl - carbohydrate ring proton 1H- 13C correlations also provide additional structural information, as shown for the 3-D DOSY-HMQC analysis of a mixture of maltotriose and lactose per-[ carbonyl- 13C]acetates.

  7. Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts.

    PubMed

    Chance, E M; Seeholzer, S H; Kobayashi, K; Williamson, J R

    1983-11-25

    Rat hearts have been perfused in vitro with 5 mM glucose and either 5 mM acetate or 1 mM pyruvate to achieve steady state conditions, followed by replacement of the acetate with 90% enriched [2-13C]acetate or pyruvate with 90% enriched [3-13C]pyruvate. The hearts were frozen different times after addition of 13C-substrate and neutralized perchloric acid extracts from three pooled hearts per time point were used to obtain high resolution proton-decoupled 13C NMR spectra at 90.55 MHz. The 13C fractional enrichment of individual carbons of different metabolites was calculated from the area of the resolved resonances after correction for nuclear Overhauser enhancement and saturation effects. A mathematical flux model of the citric acid cycle and ancillary transamination reactions was constructed with the FACSIMILE program, and used to solve unknown flux parameters with constant pool sizes by nonlinear least squares analysis of the approximately 200 simultaneous differential equations required to describe the reactions. With [2-13C] acetate as substrate, resonances and line splittings due to 13C-13C spin coupling of the C-2, C-3, and C-4 carbons of glutamate were well resolved. The half-times to reach maximum 13C enrichment were 2.6 min for glutamate C-4 and 8 min for glutamate C-2 and C-3. From these data, a well determined citric acid cycle flux of 8.3 mumol/g dry weight X min was calculated for an observed oxygen consumption of 31 mumol/g dry weight X min. With [3-13C]pyruvate as substrate, resonances of aspartate C-2 and C-3 and of alanine C-3 were well resolved in addition to those of glutamate C-2, C-3, and C-4. Nonlinear least squares fitting of these data to the model gave nonrandomly distributed residuals for the 13C fractional enrichments of glutamate C-4, suggesting an incomplete model, but a well determined cycle flux of 11.9 mumol/g dry weight X min for an oxygen uptake of 35 mumol/g dry weight X min. Our studies demonstrate the practicality of 13C NMR

  8. {sup 13}C-METHYL FORMATE: OBSERVATIONS OF A SAMPLE OF HIGH-MASS STAR-FORMING REGIONS INCLUDING ORION-KL AND SPECTROSCOPIC CHARACTERIZATION

    SciTech Connect

    Favre, Cécile; Bergin, Edwin A.; Crockett, Nathan R.; Neill, Justin L.; Carvajal, Miguel; Field, David; Jørgensen, Jes K.; Bisschop, Suzanne E.; Brouillet, Nathalie; Despois, Didier; Baudry, Alain; Kleiner, Isabelle; Margulès, Laurent; Huet, Thérèse R.; Demaison, Jean E-mail: miguel.carvajal@dfa.uhu.es

    2015-01-01

    We have surveyed a sample of massive star-forming regions located over a range of distances from the Galactic center for methyl formate, HCOOCH{sub 3}, and its isotopologues H{sup 13}COOCH{sub 3} and HCOO{sup 13}CH{sub 3}. The observations were carried out with the APEX telescope in the frequency range 283.4-287.4 GHz. Based on the APEX observations, we report tentative detections of the {sup 13}C-methyl formate isotopologue HCOO{sup 13}CH{sub 3} toward the following four massive star-forming regions: Sgr B2(N-LMH), NGC 6334 IRS 1, W51 e2, and G19.61-0.23. In addition, we have used the 1 mm ALMA science verification observations of Orion-KL and confirm the detection of the {sup 13}C-methyl formate species in Orion-KL and image its spatial distribution. Our analysis shows that the {sup 12}C/{sup 13}C isotope ratio in methyl formate toward the Orion-KL Compact Ridge and Hot Core-SW components (68.4 ± 10.1 and 71.4 ± 7.8, respectively) are, for both the {sup 13}C-methyl formate isotopologues, commensurate with the average {sup 12}C/{sup 13}C ratio of CO derived toward Orion-KL. Likewise, regarding the other sources, our results are consistent with the {sup 12}C/{sup 13}C in CO. We also report the spectroscopic characterization, which includes a complete partition function, of the complex H{sup 13}COOCH{sub 3} and HCOO{sup 13}CH{sub 3} species. New spectroscopic data for both isotopomers H{sup 13}COOCH{sub 3} and HCOO{sup 13}CH{sub 3}, presented in this study, have made it possible to measure this fundamentally important isotope ratio in a large organic molecule for the first time.

  9. Measuring glycerol turnover, gluconeogenesis from glycerol, and total gluconeogenesis with [2-13C] glycerol: role of the infusion-sampling mode.

    PubMed

    Peroni, O; Large, V; Odeon, M; Beylot, M

    1996-07-01

    Mass isotopomer distribution analysis (MIDA) of glucose during infusion of [2-13C]glycerol is a new method for measuring total gluconeogenesis (GNG). Since this method relies on calculation of the isotopic enrichment (IE) of hepatic triose phosphates (TP), the results should be independent of the sites of tracer infusion and blood sampling. Postabsorptive and starved rats were infused with [2-13C]glycerol and sampled either in the arterial-venous (A-V) or venous-arterial (V-A) modes. Blood was also sampled from the portal vein. In both postabsorptive and starved rats, glycerol turnover rate (Rt) and the percent contribution of glycerol to total glucose production were higher in the A-V mode than in the V-A mode (P < .05). Glycerol IE in portal venous blood was intermediate between IE values observed in peripheral arterial and venous blood. Its use for calculating the contribution of glycerol to glucose production reconciled the results obtained with the two infusion-sampling modes in both postabsorptive and starved rats; this contribution was increased by starvation (P < .01). In postabsorptive rats, total GNG calculated from MIDA of glucose accounted for approximately 50% of glucose production whatever the infusion-sampling mode (A-V, 48.8% +/- 4.7%; V-A, 52.2% +/- 3.9%). This contribution increased to 90% in starved rats, again, with no difference between A-V (95.2% +/- 1.8%) and V-A (89.2% +/- 1.3%) modes. In conclusion, during infusion of [2-13C]glycerol, total GNG measured from MIDA of glucose is independent of the infusion-sampling mode, contrary to calculations of Rt and GNG from glycerol. Measurement of glycerol IE in portal venous blood reconciles the results obtained with the two modes with respect to the contribution of glycerol to GNG. PMID:8692028

  10. Multi-isotope labelling of organic matter by diffusion of 2H/18O-H2O vapour and 13C-CO2 into the leaves and its distribution within the plant

    NASA Astrophysics Data System (ADS)

    Studer, M. S.; Siegwolf, R. T. W.; Leuenberger, M.; Abiven, S.

    2015-03-01

    Isotope labelling is a powerful tool to study elemental cycling within terrestrial ecosystems. Here we describe a new multi-isotope technique to label organic matter (OM). We exposed poplars (Populus deltoides × nigra) for 14 days to an atmosphere enriched in 13CO2 and depleted in 2H218O. After 1 week, the water-soluble leaf OM (δ13C = 1346 ± 162‰) and the leaf water were strongly labelled (δ18O = -63 ± 8, δ2H = -156 ± 15‰). The leaf water isotopic composition was between the atmospheric and stem water, indicating a considerable back-diffusion of vapour into the leaves (58-69%) in the opposite direction to the net transpiration flow. The atomic ratios of the labels recovered (18O/13C, 2H/13C) were 2-4 times higher in leaves than in the stems and roots. This could be an indication of the synthesis of more condensed compounds in roots and stems (e.g. lignin vs. cellulose) or might be the result of O and H exchange and fractionation processes during phloem transport and biosynthesis. We demonstrate that the three major OM elements (C, O, H) can be labelled and traced simultaneously within the plant. This approach could be of interdisciplinary interest in the fields of plant physiology, palaeoclimatic reconstruction or soil science.

  11. 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

  12. 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

  13. 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. PMID:25991647

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

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash; Lorey, Daniel R., II

    2007-02-01

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

  15. 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

  16. UV-laser-based microscopic dissection of tree rings - a novel sampling tool for δ(13) C and δ(18) O studies.

    PubMed

    Schollaen, Karina; Heinrich, Ingo; Helle, Gerhard

    2014-02-01

    UV-laser-based microscopic systems were utilized to dissect and sample organic tissue for stable isotope measurements from thin wood cross-sections. We tested UV-laser-based microscopic tissue dissection in practice for high-resolution isotopic analyses (δ(13) C/δ(18) O) on thin cross-sections from different tree species. The method allows serial isolation of tissue of any shape and from millimetre down to micrometre scales. On-screen pre-defined areas of interest were automatically dissected and collected for mass spectrometric analysis. Three examples of high-resolution isotopic analyses revealed that: in comparison to δ(13) C of xylem cells, woody ray parenchyma of deciduous trees have the same year-to-year variability, but reveal offsets that are opposite in sign depending on whether wholewood or cellulose is considered; high-resolution tree-ring δ(18) O profiles of Indonesian teak reflect monsoonal rainfall patterns and are sensitive to rainfall extremes caused by ENSO; and seasonal moisture signals in intra-tree-ring δ(18) O of white pine are weighted by nonlinear intra-annual growth dynamics. The applications demonstrate that the use of UV-laser-based microscopic dissection allows for sampling plant tissue at ultrahigh resolution and unprecedented precision. This new technique facilitates sampling for stable isotope analysis of anatomical plant traits like combined tree eco-physiological, wood anatomical and dendroclimatological studies. PMID:24219751

  17. 13C NMR Metabolomics: INADEQUATE Network Analysis

    PubMed Central

    Clendinen, Chaevien S.; Pasquel, Christian; Ajredini, Ramadan; Edison, Arthur S.

    2015-01-01

    The many advantages of 13C NMR are often overshadowed by its intrinsically low sensitivity. Given that carbon makes up the backbone of most biologically relevant molecules, 13C NMR offers a straightforward measurement of these compounds. Two-dimensional 13C-13C correlation experiments like INADEQUATE (incredible natural abundance double quantum transfer experiment) are ideal for the structural elucidation of natural products and have great but untapped potential for metabolomics analysis. We demonstrate a new and semi-automated approach called INETA (INADEQUATE network analysis) for the untargeted analysis of INADEQUATE datasets using an in silico INADEQUATE database. We demonstrate this approach using isotopically labeled Caenorhabditis elegans mixtures. PMID:25932900

  18. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Register citations affecting appendix L, see the List of CFR Sections Affected, which appears in the... Part 305—Sample Labels ER29AU07.122 PROTOTYPE LABEL 1 ER29AU07.123 PROTOTYPE LABEL 2 ER29AU07.124 PROTOTYPE LABEL 3 ER29AU07.125 PROTOTYPE LABEL 4 ER29AU07.126 SAMPLE LABEL 1 ER29AU07.127 SAMPLE LABEL...

  19. 19 CFR 12.22 - Labels; samples.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Labels; samples. 12.22 Section 12.22 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Viruses, Serums, Toxins, Antitoxins, and Analogous Products for...

  20. 19 CFR 12.22 - Labels; samples.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Labels; samples. 12.22 Section 12.22 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Viruses, Serums, Toxins, Antitoxins, and Analogous Products for...

  1. 19 CFR 12.22 - Labels; samples.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Labels; samples. 12.22 Section 12.22 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Viruses, Serums, Toxins, Antitoxins, and Analogous Products for...

  2. Synthesis and application of (13)C-labeled 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI), an immunosuppressant observed in caramel food colorings.

    PubMed

    Elsinghorst, Paul W; Raters, Marion; Dingel, Anna; Fischer, Jochen; Matissek, Reinhard

    2013-08-01

    2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is a minor toxic contaminant observed in caramel food colorings and was shown to exert immunosuppressant activity when fed to rodents. Because of this toxicity, maximum levels of THI in caramel food colorings have been defined by international and European authorities. Several reports of THI analysis using external standardization have been published for liquid foods such as beers and soft drinks. However, no suitable internal standard has yet been described allowing THI analysis in more complex samples. In this paper we describe the preparation of a labeled [(13)C6]THI analogue and its application for the successful validation of the first stable isotope dilution assay (SIDA) of THI in caramel food colorings. A brief survey of THI levels in commercially available caramel class III (E 150c) and IV (E 150d) food colorings is also included, corroborating that THI occurs only in caramel class III food colorings. PMID:23866086

  3. Photobioreactor design for isotopic non-stationary 13C-metabolic flux analysis (INST 13C-MFA) under photoautotrophic conditions.

    PubMed

    Martzolff, Arnaud; Cahoreau, Edern; Cogne, Guillaume; Peyriga, Lindsay; Portais, Jean-Charles; Dechandol, Emmanuel; Le Grand, Fabienne; Massou, Stéphane; Gonçalves, Olivier; Pruvost, Jérémy; Legrand, Jack

    2012-12-01

    Adaptive metabolic behavior of photoautotrophic microorganisms toward genetic and environmental perturbations can be interpreted in a quantitative depiction of carbon flow through a biochemical reaction network using isotopic non-stationary (13) C-metabolic flux analysis (INST (13) C-MFA). To evaluate (13) C-metabolic flux maps for Chlamydomonas reinhardtii, an original experimental framework was designed allowing rapid, reliable collection of high-quality isotopomer data against time. It involved (i) a short-time (13) C labeling injection device based on mixing control in a torus-shaped photobioreactor with plug-flow hydrodynamics allowing a sudden step-change in the (13) C proportion in the substrate feed and (ii) a rapid sampling procedure using an automatic fast filtration method coupled to a manual rapid liquid nitrogen quenching step. (13) C-substrate labeling enrichment was controlled through the total dissolved inorganic carbon concentration in the pulsed solution. First results were obtained from steady-state continuous culture measurements allowing the characterization of the kinetics of label incorporation into light-limited growing cells cultivated in a photobioreactor operating at the maximal biomass productivity for an incident photon flux density of 200 µmol m(-2) s(-1). (13)C label incorporation was measured for 21 intracellular metabolites using IC-MS/MS in 58 samples collected across a labeling experiment duration of 7 min. The fastest labeling rate was observed for 2/3-phosphoglycerate with an apparent isotopic stationary state reached after 300 s. The labeling rate was consistent with the optimized mixing time of about 4.9 s inside the reactor and the shortest reliable sampling period assessed at 5 s. PMID:22688667

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

    PubMed

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

    2015-11-01

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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. In folio respiratory fluxomics revealed by 13C isotopic labeling and H/D isotope effects highlight the noncyclic nature of the tricarboxylic acid "cycle" in illuminated leaves.

    PubMed

    Tcherkez, Guillaume; Mahé, Aline; Gauthier, Paul; Mauve, Caroline; Gout, Elizabeth; Bligny, Richard; Cornic, Gabriel; Hodges, Michael

    2009-10-01

    While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, (13)C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA "cycle" does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation. PMID:19675152

  11. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Labeling of samples. 301.33 Section 301.33 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are...

  12. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Labeling of samples. 301.33 Section 301.33 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are...

  13. Carbonation of C–S–H and C–A–S–H samples studied by {sup 13}C, {sup 27}Al and {sup 29}Si MAS NMR spectroscopy

    SciTech Connect

    Sevelsted, Tine F.; Skibsted, Jørgen

    2015-05-15

    Synthesized calcium silicate hydrate (C–S–H) samples with Ca/Si ratios of 0.66, 1.0, and 1.5 have been exposed to atmospheric CO{sub 2} at room temperature and high relative humidity and studied after one to 12 weeks. {sup 29}Si NMR reveals that the decomposition of C–S–H caused by carbonation involves two steps and that the decomposition rate decreases with increasing Ca/Si ratio. The first step is a gradual decalcification of the C–S–H where calcium is removed from the interlayer and defect sites in the silicate chains until Ca/Si = 0.67 is reached, ideally corresponding to infinite silicate chains. In the seconds step, calcium from the principal layers is consumed, resulting in the final decomposition of the C–S–H and the formation of an amorphous silica phase composed of Q{sup 3} and Q{sup 4} silicate tetrahedra. The amount of solid carbonates and of carbonate ions in a hydrous environment increases with increasing Ca/Si ratio for the C–S–H, as shown by {sup 13}C NMR. For C–A–S–H samples with Ca/Si = 1.0 and 1.5, {sup 27}Al NMR demonstrates that all aluminium sites associated with the C–S–H are consumed during the carbonation reactions and incorporated mainly as tetrahedral Al(–OSi){sub 4} units in the amorphous silica phase. A small amount of penta-coordinated Al sites has also been identified in the silica phase.

  14. Improvement of the inverse-gated-decoupling sequence for a faster quantitative analysis of various samples by 13C NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Giraudeau, Patrick; Baguet, Evelyne

    2006-05-01

    The inverse-gated-decoupling sequence enables quantitative 1H decoupled 13C spectra to be obtained. We modified this sequence so as to obtain the same result in less time for molecules containing carbons with various relaxation properties. For that, we determined the optimal 13C longitudinal-magnetization initial value for a faster relaxation while 1H decoupler is stopped. This value can be calculated precisely via the nuclear Overhauser effects, the longitudinal relaxation times, together with the determination of the relaxation rate constants of carbons while 1H are out of equilibrium. A supplementary delay of 1H decoupling and/or a series of selective pulses applied at the beginning of the recovery delay allow an acceleration of 13C longitudinal relaxation. We applied this method to the molecule of vanillin. The simultaneous quantification of all carbons was carried out with a recovery delay divided by two compared to the usual sequence.

  15. Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C-valine isotopic ratios in complex biological samples.

    PubMed

    Godin, Jean-Philippe; Breuillé, Denis; Obled, Christiane; Papet, Isabelle; Schierbeek, Henk; Hopfgartner, Gérard; Fay, Laurent-Bernard

    2008-10-01

    On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that

  16. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Editorial Note: For Federal Register citations affecting appendix L, see the List of CFR Sections Affected... amendment published at 75 FR 49819, Aug. 16, 2010. ER29AU07.122 PROTOTYPE LABEL 1 ER29AU07.123 PROTOTYPE LABEL 2 ER29AU07.124 PROTOTYPE LABEL 3 ER29AU07.125 PROTOTYPE LABEL 4 ER29AU07.126 SAMPLE LABEL...

  17. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... For Federal Register citations affecting appendix L, see the List of CFR Sections Affected, which... Part 305—Sample Labels ER29AU07.122 PROTOTYPE LABEL 1 ER29AU07.123 PROTOTYPE LABEL 2 ER29AU07.124 PROTOTYPE LABEL 3 ER29AU07.125 PROTOTYPE LABEL 4 ER21DE11.065 ER21DE11.066 ER21DE11.067...

  18. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... the List of CFR Sections Affected, which appears in the Finding Aids section of the printed volume and... Part 305—Sample Labels ER29AU07.122 PROTOTYPE LABEL 1 ER29AU07.123 PROTOTYPE LABEL 2 ER29AU07.124 PROTOTYPE LABEL 3 ER29AU07.125 PROTOTYPE LABEL 4 ER21DE11.065 ER21DE11.066 ER21DE11.067...

  19. 1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies with a diverse array of 22 condensed tannin (CT) fractions from 9 plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans flavan-3-ol ratios can be appraised by 1H-13C HSQC NMR. The method was developed from fractions containing 44 to ~100% CT, PC/PD ratios ranging f...

  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. ¹³C labelled internal standards--a solution to minimize ion suppression effects in liquid chromatography-tandem mass spectrometry analyses of drugs in biological samples?

    PubMed

    Berg, Thomas; Strand, Dag Helge

    2011-12-30

    Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is frequently used to identify and quantify drugs in human biological samples due to the high selectivity and sensitivity of this technique. However, ion suppression effects caused by co-eluting compounds: drugs, metabolites, matrix components, impurities and degradation products, are a major concern. Stable isotope labelled internal standards (SIL ISs), usually deuterium ((2)H) labelled, are often used to compensate for these effects. In many LC separations the retention times of (2)H labelled ISs and their analogues will differ. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is increasingly being used for bio-analysis. With the better chromatographic resolution provided with sub 2 μm particles, larger separation between analytes and their (2)H labelled analogues can be expected, which might reduce the benefits of the SIL IS. There is a greater difference in physico-chemical properties between hydrogen isotopes than between isotopes of other elements. (13)C, (15)N and (18)O labelled ISs are more similar to their analytes than (2)H labelled ISs and thereby expected to behave more similarly in chromatographic separations. In this study we have investigated the use of (13)C and (2)H labelled ISs for the determination of amphetamine and methamphetamine by UPLC-MS/MS. The (13)C labelled ISs were co eluting with their analytes under different chromatographic conditions while the (2)H labelled ISs and their analytes were slightly separated. An improved ability to compensate for ion suppression effects were observed when the (13)C labelled ISs were used. Furthermore, an UPLC-MS/MS method for determination of amphetamine and methamphetamine in urine using (13)C labelled ISs has been developed and validated. Unfortunately, there are few (13)C labelled ISs commercial available today. If more (13)C labelled ISs become commercial available they may well be the coming solution to minimize

  2. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples,...

  3. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples,...

  4. 16 CFR 301.33 - Labeling of samples.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER FUR PRODUCTS LABELING ACT Regulations § 301.33 Labeling of samples. Where samples of furs or fur products subject to the act are used to promote or effect sales of fur products, said samples,...

  5. 27 CFR 20.253 - Labels for samples.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Labels for samples. 20.253 Section 20.253 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Denatured Spirits § 20.253 Labels for samples. When a sample of specially denatured spirits is...

  6. 27 CFR 20.253 - Labels for samples.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Labels for samples. 20.253 Section 20.253 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Denatured Spirits § 20.253 Labels for samples. When a sample of specially denatured spirits is...

  7. 27 CFR 20.253 - Labels for samples.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Labels for samples. 20.253 Section 20.253 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Denatured Spirits § 20.253 Labels for samples. When a sample of specially denatured spirits is...

  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. /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.

  10. Towards hyperpolarized 13C-succinate imaging of brain cancer

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Pratip; Chekmenev, Eduard Y.; Perman, William H.; Harris, Kent C.; Lin, Alexander P.; Norton, Valerie A.; Tan, Chou T.; Ross, Brian D.; Weitekamp, Daniel P.

    2007-05-01

    We describe a novel 13C enriched precursor molecule, sodium 1- 13C acetylenedicarboxylate, which after hydrogenation by PASADENA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized 13C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized 13C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized 13C sodium succinate, contained significant concentrations of the injected substrate, 13C sodium succinate, together with 13C maleate and succinate metabolites 1- 13C-glutamate, 5- 13C-glutamate, 1- 13C-glutamine and 5- 13C-glutamine. The 13C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood-brain barrier. These ex vivo results indicate that hyperpolarized 13C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in 13C MR spectral-spatial images.