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Sample records for selectively 13c labeled

  1. Site-selective 13C labeling of proteins using erythrose

    International Nuclear Information System (INIS)

    Weininger, Ulrich

    2017-01-01

    NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13 C and/or 1 H, which is achieved in the most general way by using site-selectively 13 C-enriched glucose (1- and 2- 13 C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13 C-enriched erythrose (1-, 2-, 3- and 4- 13 C) as a suitable precursor for 13 C labeled aromatic side chains. We quantify 13 C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13 C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13 C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13 C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

  2. Site-selective {sup 13}C labeling of proteins using erythrose

    Energy Technology Data Exchange (ETDEWEB)

    Weininger, Ulrich, E-mail: ulrich.weininger@physik.uni-halle.de [Lund University, Department of Biophysical Chemistry, Center for Molecular Protein Science (Sweden)

    2017-03-15

    NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with {sup 13}C and/or {sup 1}H, which is achieved in the most general way by using site-selectively {sup 13}C-enriched glucose (1- and 2-{sup 13}C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively {sup 13}C-enriched erythrose (1-, 2-, 3- and 4-{sup 13}C) as a suitable precursor for {sup 13}C labeled aromatic side chains. We quantify {sup 13}C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the {sup 13}C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated {sup 13}C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective {sup 13}C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

  3. Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations

    International Nuclear Information System (INIS)

    Hong, M.; Jakes, K.

    1999-01-01

    The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated

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

    Energy Technology Data Exchange (ETDEWEB)

    SantaLucia, J. Jr.; Shen, L.X.; Lewis, H.; Cai, Z.; Tinoci, I. Jr. [Univ. of California, Berkeley, CA (United States)

    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).

  5. Radiation oxidation of polypropylene: A solid-state 13C NMR study using selective isotopic labeling

    International Nuclear Information System (INIS)

    Mowery, Daniel M.; Assink, Roger A.; Derzon, Dora K.; Klamo, Sara B.; Bernstein, Robert; Clough, Roger L.

    2007-01-01

    Polypropylene samples, in which the three different carbon atoms along the chain were selectively labeled with carbon-13, were subjected to radiation under inert and air atmospheres, and to post-irradiation exposure in air at various temperatures. By using solid-state 13 C NMR measurements at room temperature, we have been able to identify and quantify the oxidation products. The isotopic labeling provides insight into chemical reaction mechanisms, since oxidation products can be traced back to their positions of origin on the macromolecule. The major products include peroxides and alcohols, both formed at tertiary carbon sites along the chain. Other products include methyl ketones, acids, esters, peresters, and hemiketals formed from reaction at the tertiary carbon, together with in-chain ketones and esters from reaction at the secondary chain carbon. No evidence is found of products arising from reactions at the methyl side chain. Significant temperature-dependent differences are apparent; for example much higher yields of chain-end methyl ketones, which are the indicator product of chain scission, are generated for both elevated temperature irradiation and for post-irradiation treatment at elevated temperatures. Time-dependent plots of yields of the various oxidation products have been obtained under a wide range of conditions, including the post-irradiation oxidation of a sample at room temperature in air that has been monitored for 2 years. Radiation-oxidation products of polypropylene are contrasted to products measured for 13 C-labeled polyethylene in an earlier investigation: the peroxides formed in irradiated polypropylene are remarkably longer lived, the non-peroxidic products are significantly different, and the overall ratios of oxidation products in polypropylene change relatively little as a function of the extent of oxidation

  6. Perdeuteration and methyl-selective 1H, 13C-labeling by using a Kluyveromyces lactis expression system

    International Nuclear Information System (INIS)

    Miyazawa-Onami, Mayumi; Takeuchi, Koh; Takano, Toshiaki; Sugiki, Toshihiko; Shimada, Ichio; Takahashi, Hideo

    2013-01-01

    The production of stable isotope-labeled proteins is critical in structural analyses of large molecular weight proteins using NMR. Although prokaryotic expression systems using Escherichia coli have been widely used for this purpose, yeast strains have also been useful for the expression of functional eukaryotic proteins. Recently, we reported a cost-effective stable isotope-labeled protein expression using the hemiascomycete yeast Kluyveromyces lactis (K. lactis), which allow us to express exogenous proteins at costs comparable to prokaryotic expression systems. Here, we report the successful production of highly deuterated (>90 %) protein in the K. lactis system. We also examined the methyl-selective 1 H, 13 C-labeling of Ile, Leu, and Val residues using commonly used amino acid precursors. The efficiency of 1 H- 13 C-incorporation varied significantly based on the amino acid. Although a high level of 1 H- 13 C-incorporation was observed for the Ile δ1 position, 1 H, 13 C-labeling rates of Val and Leu methyl groups were limited due to the mitochondrial localization of enzymes involved in amino acid biosynthesis and the lack of transporters for α-ketoisovalerate in the mitochondrial membrane. In line with this notion, the co-expression with branched-chain-amino-acid aminotransferase in the cytosol significantly improved the incorporation rates of amino acid precursors. Although it would be less cost-effective, addition of 13 C-labeled valine can circumvent problems associated with precursors and achieve high level 1 H, 13 C-labeling of Val and Leu. Taken together, the K. lactis system would be a good alternative for expressing large eukaryotic proteins that need deuteration and/or the methyl-selective 1 H, 13 C-labeling for the sensitive detection of NMR resonances

  7. Synthesis of selectively 13C-labelled benzoic acid for nuclear magnetic resonance spectroscopic measurement of glycine conjugation activity

    International Nuclear Information System (INIS)

    Akira, Kazuki; Hasegawa, Hiroshi; Baba, Shigeo

    1995-01-01

    The synthesis of [4- 13 C]benzoic acid (BA) labelled in a single protonated carbon, for use as a probe to measure glycine conjugation activity by nuclear magnetic resonance (NMR) spectroscopy, has been reported. The labelled compound was prepared by a seven-step synthetic scheme on a relatively small scale using [2- 13 C] acetone as the source of label in overall yield of 16%. The usefulness of [4- 13 C]BA was demonstrated by the NMR spectroscopic monitoring of urinary excretion of [4- 13 C]hippuric acid in the rat administered with the labelled BA. (Author)

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

    Energy Technology Data Exchange (ETDEWEB)

    LeMaster, D.M. [Northwestern Univ., Evanston, IL (United States)

    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.

  9. A facile synthesis of δ-aminolevulinic acid (ALA) regio-selectively labeled with 13C and direct observation of enzymatic transformation from ALA to porphobilinogen (PBG)

    International Nuclear Information System (INIS)

    Kurumaya, Katsuyuki; Okazaki, Takeo; Seido, Nobuo; Akasaka, Yuzuru; Kawajiri, Yoshiki; Kajiwara, Masahiro; Kondo, Masao

    1989-01-01

    δ-Aminolevulinic acid (ALA), labeled with 13 C at position 1, 2, 3, 4, or 5, was synthesized from 13 C-labeled glycine, Meldrum's acid, or bromoacetate. The latter compounds were prepared from 13 C-sodium acetate or 13 C-acetic acid. Enzymatic transformation from ALA to porphobilinogen (PBG) was directly observed by 13 C-NMR. (author)

  10. A facile synthesis of. delta. -aminolevulinic acid (ALA) regio-selectively labeled with sup 13 C and direct observation of enzymatic transformation from ALA to porphobilinogen (PBG)

    Energy Technology Data Exchange (ETDEWEB)

    Kurumaya, Katsuyuki; Okazaki, Takeo; Seido, Nobuo; Akasaka, Yuzuru; Kawajiri, Yoshiki; Kajiwara, Masahiro (Meiji College of Pharmacy, Tokyo (Japan)); Kondo, Masao (Institute of Public Health, Tokyo (Japan))

    1989-02-01

    {delta}-Aminolevulinic acid (ALA), labeled with {sup 13}C at position 1, 2, 3, 4, or 5, was synthesized from {sup 13}C-labeled glycine, Meldrum's acid, or bromoacetate. The latter compounds were prepared from {sup 13}C-sodium acetate or {sup 13}C-acetic acid. Enzymatic transformation from ALA to porphobilinogen (PBG) was directly observed by {sup 13}C-NMR. (author).

  11. {sup 15}N and {sup 13}C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated, selectively [{sup 1}H,{sup 13}C]-labeled proteins

    Energy Technology Data Exchange (ETDEWEB)

    Rossi, Paolo, E-mail: rossip@umn.edu; Xia, Youlin; Khanra, Nandish; Veglia, Gianluigi, E-mail: vegli001@umn.edu; Kalodimos, Charalampos G., E-mail: ckalodim@umn.edu [University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics (United States)

    2016-12-15

    The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and {sup 15}N, methyl labeled samples in H{sub 2}O. The experiments benefit from a combination of selective T{sub 1} relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear {sup 15}N,{sup 13}C-edited, or with diagonal-free {sup 13}C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.

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

    Science.gov (United States)

    Fontana, Carolina; Kovacs, Helena; Widmalm, Göran

    2014-06-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    Highlights: • 13 C labeled testosterone can be used to adjust the isotope ratio of testosterone. • The novel testosterone cannot be detected by the regular IRMS method in doping test. • A method was explored to remove the labeled 13 C. • The established method can be used to detect the manipulated testosterone. - Abstract: 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 C Andro–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 C Andro–ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ 13 C Andro–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

  14. 13C metabolic flux analysis: optimal design of isotopic labeling experiments.

    Science.gov (United States)

    Antoniewicz, Maciek R

    2013-12-01

    Measuring fluxes by 13C metabolic flux analysis (13C-MFA) has become a key activity in chemical and pharmaceutical biotechnology. Optimal design of isotopic labeling experiments is of central importance to 13C-MFA as it determines the precision with which fluxes can be estimated. Traditional methods for selecting isotopic tracers and labeling measurements did not fully utilize the power of 13C-MFA. Recently, new approaches were developed for optimal design of isotopic labeling experiments based on parallel labeling experiments and algorithms for rational selection of tracers. In addition, advanced isotopic labeling measurements were developed based on tandem mass spectrometry. Combined, these approaches can dramatically improve the quality of 13C-MFA results with important applications in metabolic engineering and biotechnology. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Application of 13C-labeling and 13C-13C COSY NMR experiments in the structure determination of a microbial natural product.

    Science.gov (United States)

    Kwon, Yun; Park, Sunghyouk; Shin, Jongheon; Oh, Dong-Chan

    2014-08-01

    The elucidation of the structures of complex natural products bearing many quaternary carbons remains challenging, even in this advanced spectroscopic era. (13)C-(13)C COSY NMR spectroscopy shows direct couplings between (13)C and (13)C, which comprise the backbone of a natural product. Thus, this type of experiment is particularly useful for natural products bearing consecutive quaternary carbons. However, the low sensitivity of (13)C-based NMR experiments, due to the low natural abundance of the (13)C nucleus, is problematic when applying these techniques. Our efforts in the (13)C labeling of a microbial natural product, cyclopiazonic acid (1), by feeding (13)C-labeled glucose to the fungal culture, enabled us to acquire (13)C-(13)C COSY NMR spectra on a milligram scale that clearly show the carbon backbone of the compound. This is the first application of (13)C-(13)C COSY NMR experiments for a natural product. The results suggest that (13)C-(13)C COSY NMR spectroscopy can be routinely used for the structure determination of microbial natural products by (13)C-enrichment of a compound with (13)C-glucose.

  17. Overexpression of a homogeneous oligosaccharide with {sup 13}C labeling by genetically engineered yeast strain

    Energy Technology Data Exchange (ETDEWEB)

    Kamiya, Yukiko; Yamamoto, Sayoko [National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience and Institute for Molecular Science (Japan); Chiba, Yasunori; Jigami, Yoshifumi [National Institute of Advanced Industrial Science and Technology, Research Center for Medical Glycoscience (Japan); Kato, Koichi, E-mail: kkatonmr@ims.ac.jp [National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience and Institute for Molecular Science (Japan)

    2011-08-15

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

  18. Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

    International Nuclear Information System (INIS)

    Lee, Andrew L.; Urbauer, Jeffrey L.; Wand, A. Joshua

    1997-01-01

    Selective incorporation of 13 C into the methyl groups of protein side chains is described as a means for simplifying the measurement and interpretation of 13 C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3- 13 C, 99%) as the sole carbon source in the growth media for protein overexpression in E. coli. This improved labeling scheme increases the sensitivity of the relaxation experiments by approximately fivefold when compared to randomly fractionally 13 C-labeled protein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost

  19. Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans

    International Nuclear Information System (INIS)

    Yamaguchi, Yoshiki; Kato, Koichi; Shindo, Mitsuru; Aoki, Shin; Furusho, Kumiko; Koga, Kenji; Takahashi, Noriko; Arata, Yoji; Shimada, Ichio

    1998-01-01

    A systematic method for 13 C labeling of the glycan of immunoglobulin G for NMR study has been developed. A mouse immunoglobulin of subclass IgG2b has been used for the experiment. On the basis of chemical shift and linewidth data, it has been concluded that (1) the mobility of the carbohydrate chain in IgG2b is comparable to that of the backbone polypeptide chain with the exception of the galactose residue at the nonreducing end of the Man α 1-3 branch, which is extremely mobile and (2) agalactosylation does not induce any significant change in the mobility. The results obtained indicate that even in the agalactosyl form the glycans are buried in the protein. Biological significance of the NMR results obtained is also briefly discussed

  20. Synthesis of ring-13C-labelled and ring-demethylated retinals

    International Nuclear Information System (INIS)

    Courtin, J.M.L.

    1988-01-01

    Efficient synthetic schemes are described for the preparation of the required mono- and di- 13 C labelled retinals based on simple 13 C labelled starting materials. Results from solid-state 13 C-NMR spectroscopic studies of the various ring- 13 C labelled bacteriorhodopsins and rhodopsins are discussed. 404 refs.; 74 figs.; 16 tabs

  1. Synthesis of {sup 14}C-labeled levamisole and {sup 13}C-labeled tetramisole

    Energy Technology Data Exchange (ETDEWEB)

    Feil, V.J. [US Department of Agriculture, Agricultural Research Service, Biosciences Research Lab., Fargo, ND (United States)

    1996-12-01

    The syntheses of {sup 14}C-ring labeled levamisole ([-]-2,3,5,6-tetrahydro-6-phenyl [{sup 14}C]-UL imidazo[2,1-b]thiazole) from acetophenone-ring-UL-{sup 14}C in 5 steps plus resolution with a 7.5% overall yield, and {sup 13}C{sub 6}-ring labeled tetramisole ([{+-}]-2,3,5,6-tetrahydro-6-phenyl [{sup 13}C{sub 6}]imidazo[2,1-b]thiazole) from benzene-{sup 13}C{sub 6} in 6 steps with a 9.0% overall yield are described. (author).

  2. Synthesis and purification of 13C labelled xanthine derivatives

    International Nuclear Information System (INIS)

    Boukraa, M.S.; Deruaz, D.; Bannier, A.; Desage, M.; Brazier, J.L.

    1995-01-01

    3-[Methyl- 13 )C]xanthine, 7-[Methyl- 13 )C]xanthine, 1,3-[Dimethyl- 13 )C 2 ]xanthine (theophylline-1,3-[ 13 )CH 3 ] 2 ), 1,7-[Dimethyl- 13 )C 2 ]xanthine (paraxanthine-1,7[ 13 )CH 3 ] 2 ), and 3,7-[Dimethyl- 13 )C 2 ]xanthine (theobromine-3,7-[ 13 )CH 3 ] 2 were synthesized by nucleophilic substitution reaction(SN 2 ) from xanthine (X) and iodomethane-[ 13 C]. The 3-isobutylparaxanthine-7-[ 13 CH 3 ] was prepared from 3-isobutyl-1-methylxanthine (IBMX). The compounds were purified by reverse phase semipreparative liquid chromatography and their chemical structure and purity verified by GC-MS. (Author)

  3. IRMS detection of testosterone manipulated with {sup 13}C labeled standards in human urine by removing the labeled {sup 13}C

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jingzhu, E-mail: wangjingzhu@chinada.cn [National Anti-Doping Laboratory, China Anti-Doping Agency, Beijing (China); Yang, Rui [Sport Science College, Beijing Sport University Beijing, Beijing (China); Yang, Wenning [School of Pharmacy, Beijing University of Chinese Medicine, Beijing (China); Liu, Xin; Xing, Yanyi; Xu, Youxuan [National Anti-Doping Laboratory, China Anti-Doping Agency, Beijing (China)

    2014-12-10

    Highlights: • {sup 13}C labeled testosterone can be used to adjust the isotope ratio of testosterone. • The novel testosterone cannot be detected by the regular IRMS method in doping test. • A method was explored to remove the labeled {sup 13}C. • The established method can be used to detect the manipulated testosterone. - Abstract: Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ{sup 13}C value). However, {sup 13}C labeled standards can be used to control the δ{sup 13}C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the {sup 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{sup 13}C values between Andro and ANAD (Δδ{sup 13}C{sub Andro–ANAD}, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different {sup 13}C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ{sup 13}C{sub Andro–ANAD} post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ{sup 13}C{sub Andro–ANAD} for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-{sup 13}C labeled standards.

  4. Synthesis of 13C and 2H labelled retinals: spectroscopic investigations on isotopically labelled rhodopsin and bacteriorhodopsin

    International Nuclear Information System (INIS)

    Pardoen, J.A.

    1986-01-01

    In order to develop probes of the structure of chromophores, the author introduces isotopic modifications at specific chromophoric positions as structural probes. To obtain bacteriorhodopsin, rhodopsin and their photoproducts labelled in the chromophore at selected positions, bacterioopsin and opsin were reacted with the appropriate labelled a11-trans and 11-cis retinals. The author describes the synthesis of a11-trans retinal selectively 13 C labelled at different positions. The characterization of these labelled a11-trans retinals by mass spectrometry, 300 MHz 1 H NMR and 75 MHz 13 C NMR spectroscopy is given. The photochemical preparation and isolation of the pure 9-, 11- and 13-cis forms is described in the experimental part. (Auth.)

  5. Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

    International Nuclear Information System (INIS)

    Jacob, Jaison; Louis, John M.; Nesheiwat, Issa; Torchia, Dennis A.

    2002-01-01

    Analysis of 2D [ 13 C, 1 H]-HSQC spectra of biosynthetic fractionally 13 C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional 13 C labeling yields aromatic rings in which some of the 13 C- 13 C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the δ-, ε- and ζ-carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the 13 C constant-time period in 2D [ 13 C, 1 H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic 13 C CSA and 13 C- 1 H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution 13 C constant-time spectra with good sensitivity

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

    International Nuclear Information System (INIS)

    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 13 C labeling in such investigations can be illustrated by the results on suspensions of the yeast, Candida utilis

  7. Synthesis of [sup 13]C warfarin labelled at the hemiketal carbon, and its resolution

    Energy Technology Data Exchange (ETDEWEB)

    Savell, V.H. Jr.; Valente, E.J. (Mississippi College, Clinton. MS (United States). Dept. of Chemistry); Eggleston, D.S. (Smith, Kline and French Labs., King of Prussia, PA (United States). Physical and Structural Chemistry)

    1989-06-01

    Warfarin (cyclic hemiketal form: 2-hydroxy-2-methyl-4-phenyl-3,4-dihydro-2H,5H-pyrano[3,2-c][1]benz opyran-5-one) is labeled with 98+% [sup 13]C at the anomeric carbon (C2) and resolved into its enantiomers. Acetone-2-[sup 13]C(98.6%) condenses with benzaldehyde in aqueous base to produce 4-phenyl-3-buten-2-one-2-[sup 13]C(98+%). Michael-type addition of this to 4-hydroxycoumarin in methanol produces the labeled diastereomeric warfarin methyl ketals which on deprotection form racemic warfarin-2-[sup 13]C(98+%). Classical resolution of labeled warfarin with quinidine produces partly resolved (S)-(-)-warfarin-2-[sup 13]C(98+%). Labeled warfarin is a suitable probe for warfarin configuration for which three distinct isomeric forms are known. (Author).

  8. Synthesis of 13C warfarin labelled at the hemiketal carbon, and its resolution

    International Nuclear Information System (INIS)

    Savell, V.H. Jr.; Valente, E.J.; Eggleston, D.S.

    1989-01-01

    Warfarin (cyclic hemiketal form: 2-hydroxy-2-methyl-4-phenyl-3,4-dihydro-2H,5H-pyrano[3,2-c][1]benz opyran-5-one) is labeled with 98+% 13 C at the anomeric carbon (C2) and resolved into its enantiomers. Acetone-2- 13 C(98.6%) condenses with benzaldehyde in aqueous base to produce 4-phenyl-3-buten-2-one-2- 13 C(98+%). Michael-type addition of this to 4-hydroxycoumarin in methanol produces the labeled diastereomeric warfarin methyl ketals which on deprotection form racemic warfarin-2- 13 C(98+%). Classical resolution of labeled warfarin with quinidine produces partly resolved (S)-(-)-warfarin-2- 13 C(98+%). Labeled warfarin is a suitable probe for warfarin configuration for which three distinct isomeric forms are known. (Author)

  9. Synthesis of C-13 labeled vitamin E, [4' a-13C]all-rac-α-tocopherol

    International Nuclear Information System (INIS)

    Urano, Shiro; Muto, Riko; Matsuo, Mitsuyoshi

    1985-01-01

    Vitamin E with a 13 C-labeled isoprenoid side chain, [4' a- 13 C]-all-rac-α-tocopherol, was synthesized by the coupling reaction of 6-4-methoxymethoxy-2-([methyl- 13 C]5-bromo-4-methyl-pent-1-yl)chroman (8) with 3,7-dimethyl-1-(thiazolin-2-yl)thio-2,6-octadiene. Compound 8 was prepared using 2-(4,4-di-ethoxycarbonylbut-1-yl)-6-methoxymethoxy-2,5,7,8-tetramethyl-chroman as a key intermediate and [ 13 C]methyl iodide as a 13 C source. The total yield of the labeled α-tocopherol based on [ 13 C]methyl iodide was 58.7%. (author)

  10. Biosynthesis of the sesquiterpene germacrene D in Solidago canadensis: 13C and (2)H labeling studies.

    Science.gov (United States)

    Steliopoulos, Panagiotis; Wüst, Matthias; Adam, Klaus-Peter; Mosandl, Armin

    2002-05-01

    The biogenetic origin of the isoprenoid building blocks of the sesquiterpene germacrene D was studied in Solidago canadensis. Feeding experiments were carried out with 1-[5,5-D(2)]deoxy-D-xylulose-5-phosphate (D(2)-DOXP), [5-13C]mevalonolactone (13C-MVL) and [1-13C]-D-glucose. The hydrodistillate of a cut shoot fed with D(2)-DOXP was investigated by enantio-MDGC-MS and the volatile fraction of a shoot supplied with 13C-MVL was examined by GC-C-IRMS. The incorporation of [1-13C]-D-glucose was analyzed by quantitative 13C NMR spectroscopy after isolation of germacrene D from the essential oil. Our labeling studies revealed that the biosynthesis of the C-15 skeleton of sesquiterpene germacrene D in Solidago canadensis proceeds predominantly via the methylerythritol phosphate pathway.

  11. Identification of degradation routes of metamitron in soil microcosms using 13C-isotope labeling.

    Science.gov (United States)

    Wang, Shizong; Miltner, Anja; Nowak, Karolina M

    2017-01-01

    Metamitron is one of the most commonly used herbicide in sugar beet and flower bulb cultures. Numerous laboratory and field studies on sorption and degradation of metamitron were performed. Detailed biodegradation studies in soil using 13 C-isotope labeling are still missing. Therefore, we aimed at providing a detailed turnover mass balance of 13 C 6 -metamitron in soil microcosms over 80 days. In the biotic system, metamitron mineralized rapidly, and 13 CO 2 finally constituted 60% of the initial 13 C 6 -metamitron equivalents. In abiotic control experiments CO 2 rose to only 7.4% of the initial 13 C 6 -metamitron equivalents. The 13 C label from 13 C 6 -metamitron was incorporated into microbial amino acids that were ultimately stabilized in the soil organic matter forming presumably harmless biogenic residues. Finally, 13 C label from 13 C 6 -metamitron was distributed between the 13 CO 2 and the 13 C-biogenic residues indicating nearly complete biodegradation. The parallel increase of 13 C-alanine, 13 C-glutamate and 13 CO 2 indicates that metamitron was initially biodegraded via the desamino-metamitron route suggesting its relevance in the growth metabolism. In later phases of biodegradation, the "Rhodococcus route" was indicated by the low 13 CO 2 evolution and the high relevance of the pyruvate pathway, which aims at biomolecule synthesis and seems to be related to starvation. This is a first report on the detailed degradation route of metamitron in soil. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Isotopic separation of 13C by selective photodissociation of formaldehyde

    International Nuclear Information System (INIS)

    Mussillon, T.

    1998-01-01

    The aim of this work is to study the feasibility of the 13 C isotopic separation by UV laser spectroscopy. The spectra of H 2 12 CO and H 2 13 CO have been recorded by a Fourier transform spectrometer between 28000 and 34000 cm -1 . From these data has been carried out a systematic study of some lines by laser spectroscopy. The selectivity measurements have been compared with the obtained enrichment factors. Thus has been revealed in a quantitative way, the importance of the isotopic re-mixture phenomena and of the selectivity loss. The best enrichment factor has been measured at 29935,56 cm -1 (band: (2,14,1)). A final percentage of 42,1 % has been obtained in a reproducible way for 13 C. The evolution of the enrichment factor has been characterized for a pressure range between 4,4 and 43 mbar. Above the radical dissociation threshold, it has not be possible to show a positive effect of NO on the enrichment factor. This negative result has been explained by a detailed kinetic study of the radical reactions (available literature). This experimental study has been completed by a bibliographic synthesis for understanding the formaldehyde photochemistry. All the processes able to influence the performance of this isotopic separation process have been gathered in this work in an exhaustive way. The radical dissociation threshold of H 2 13 CO have been calculated from molecular constants of the literature and from known thermodynamic data for H 2 12 CO. (O.M.)

  13. The synthesis of Org 3770 labelled with 3H, 13C and 14C

    International Nuclear Information System (INIS)

    Kaspersen, F.M.; Rooij, F.A.M. van; Sperling, E.G.M.; Wieringa, J.H.

    1989-01-01

    The syntheses of 1,2,3,4,10,14b-hexahydro-2-methylpyrazino[2,1-a]pyrido[2,3-c][2]benazepine (Org 3770) labelled with 3 H (and 2 H), 13 C and 14 C are described. Tritiated Org 3770 was prepared either by exchange under alkaline conditions with tritiated water or catalytic reductive dehalogenation of a chloro analogue with 3 H 2 . 13 C-labelled material was obtained in a seven-step synthesis starting from 13 C-labelled benzene whereas 14 C-Org 3770 was prepared in a three-step synthesis starting with 14 CO 2 . All labelled compounds were analyzed by TLC, HPLC, MS and NMR. (author)

  14. 13C and 31P NMR study of gluconeogenesis: utilization of 13C-labeled substrates by perfused liver from streptozotocin-diabetic and untreated rats

    International Nuclear Information System (INIS)

    Cohen, S.M.

    1987-01-01

    The metabolism of 13 C-labeled substrates was followed by 13 C and 31 P NMR in perfused liver from the streptozotocin-treated rat model of insulin-dependent diabetes. Comparison was made with perfused liver from untreated littermates, fasted either 24 or 12 h. The major routes of pyruvate metabolism were followed by a 13 C NMR approach that provided for the determination of the metabolic fate of several substances simultaneously. The rate of gluconeogenesis was 2-4-fold greater and β-hydroxybutyrate production was 50% greater in liver from the chronically diabetic rats as compared with the control groups. Large differences in the distribution of 13 C label in hepatic alanine were measured between diabetic and control groups. The biosyntheses of 13 C-labeled glutathione and N-carbamoylaspartate were monitored in time-resolved 13 C NMR spectra of perfused liver. Assignments for the resonances of glutathione and N-carbamoylaspartate were made with the aid of 13 C NMR studies of perchloric acid extracts of the freeze-clamped livers. 13 C NMR spectroscopy of the perfusates provided a convenient, rapid assay of the rate of oxidation of [2- 13 C]ethanol, the hepatic output of [2- 13 ]acetaldehyde, and the accumulation of [2- 13 C]acetate in the perfusate. By 31 P NMR spectroscopy, carbamoyl phosphate was measured in all diabetic livers and an unusual P,P'-diesterified pyrophosphate was observed in one-fourth of the diabetic livers examined. Neither of these phosphorylated metabolites was detected in control liver. Both 13 C and 31 P NMR were useful in defining changes in hepatic metabolism in experimental diabetes

  15. Quantitative evaluation of the biosynthetic pathways leading to δ-aminolevulinic acid from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus by analysis of 13C-labeled coproporphyrinogen III biosynthesized from [2-13C]glycine, [1-13C]acetate, and [2-13C]acetate using 13C NMR spectroscopy

    International Nuclear Information System (INIS)

    Katsumi Iida

    2013-01-01

    The biosynthetic pathways leading to δ-aminolevulinic acid (ALA) from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus were quantitatively evaluated by means of feeding experiments with [2- 13 C]glycine, sodium [1- 13 C]acetate, and sodium [2- 13 C]acetate, followed by analysis of the labeling patterns of coproporphyrinogen III (Copro'gen III) (biosynthesized from ALA) using 13 C NMR spectroscopy. Two biosynthetic pathways leading to ALA from glycine via the C5 pathway were identified: i.e., transformation of glycine to l-serine catalyzed by glycine hydroxymethyltransferase, and glycine synthase-catalyzed catabolism of glycine to N 5 , N 10 -methylene-tetrahydrofolic acid (THF), which reacts with another molecule of glycine to afford l-serine. l-Serine is transformed to acetyl-CoA via pyruvic acid. Acetyl-CoA enters the tricarboxylic acid cycle, affording 2-oxoglutaric acid, which in turn is transformed to l-glutamic acid. The l-glutamic acid enters the C5 pathway, affording ALA in A. hyalinus. A 13 C NMR spectroscopic comparison of the labeling patterns of Copro'gen III obtained after feeding of [2- 13 C]glycine, sodium [1- 13 C]acetate, and sodium [2- 13 C]acetate showed that [2- 13 C]glycine transformation and [2- 13 C]glycine catabolism in A. hyalinus proceed in the ratio of 52 and 48 %. The reaction of [2- 13 C]glycine and N 5 , N 10 -methylene-THF, that of glycine and N 5 , N 10 -[methylene- 13 C]methylene-THF generated from the [2- 13 C]glycine catabolism, and that of [2- 13 C]glycine and N 5 , N 10 -[methylene- 13 C]methylene-THF transformed the fed [2- 13 C]glycine to [1- 13 C]acetyl-CoA, [2- 13 C]acetyl-CoA, and [1,2- 13 C 2 ]acetyl-CoA in the ratios of 42, 37, and 21 %, respectively. These labeled acetyl-CoAs were then incorporated into ALA. Our results provide a quantitative picture of the pathways of biosynthetic transformation to ALA from glycine in A. hyalinus. (author)

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

    Science.gov (United States)

    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. Copyright © 2014 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2016-04-01

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

  18. Preparation of 2H- and 13C-labelled precursors of 2-hydroxy-1, 3-butadiene

    International Nuclear Information System (INIS)

    Turecek, F.

    1987-01-01

    2-exo-Vinylbicyclo[2.2.1]hept-5-en-2-ols, specifically labelled with 2 H at C-3 and in the vinyl group were prepared from bicyclo[2.2.1]hept-5-en-2-one in several steps. [4- 13 C]oct-1-en-3-one was prepared in five steps from 13 CO 2 . These compounds serve as precursors for the preparation of specifically labelled neutral and ionized 2-hydroxy-1, 3-butadienes. (author)

  19. Synthesis of 13C-labelled lactose for metabolic studies in subjects with gastrointestinal disorders

    International Nuclear Information System (INIS)

    Moyna, P.

    1993-01-01

    The long-range goals included development of a 13 C-labelled lactose method for measuring lactose malabsorption in patients with diarrhea. The short-term goals included assembling a nuclear magnetic resonance system and a computer system for spectra analysis. The latter results are the subject of the report. (author)

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  1. Heteronuclear 2D NMR studies on an engineered insulin monomer: Assignments and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design

    International Nuclear Information System (INIS)

    Weiss, M.A.; Hua, Qingxin; Lynch, C.S.; Shoelson, S.E.; Frank, B.H.

    1991-01-01

    Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. The authors demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10 → Asp, ProB28 → Lys, and LysB29 → Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1 H NMR studies of native human insulin and a series of three related analogues-(i) the singly substituted analogue [HisB10→Asp], (ii) the doubly substituted analogue [ProB28→Lys; LysB29→Pro], and (iii) DKP-insulin-demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2 H and 13 C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues

  2. High-throughput and homogeneous 13C-labelling of plant material for fair carbon accounting

    International Nuclear Information System (INIS)

    Slaets, J.I.F.; Resch, C.; Mayr, L.; Weltin, G.; Heiling, M.; Gruber, R.; Dercon, G.

    2016-01-01

    With growing political acknowledgement of the anthropogenic drivers and consequences of climate change, the development of carbon accounting mechanisms is essential for fair greenhouse gas emission mitigation policies. Therefore, carbon storage and emission must be accurately quantified. Plant material labelled with 13 C can be used to measure carbon storage in soil and carbon losses via CO 2 emission to the atmosphere from various cropping practices through in situ and incubation experiments.

  3. Biokinetics of 13C in the human body after oral administration of 13C-labeled glucose as an index for the biokinetics of 14C.

    Science.gov (United States)

    Masuda, Tsuyoshi; Tako, Yasuhiro; Matsushita, Kensaku; Takeda, Hiroshi; Endo, Masahiro; Nakamura, Yuji; Hisamatsu, Shun'ichi

    2016-09-01

    The retention of 13 C in the human body after oral administration of 13 C-labeled glucose was studied in three healthy volunteer subjects to estimate the 50 year cumulative body burden for 13 C as an index of the committed dose of the radioisotope 14 C. After administration of 13 C-labeled glucose, the volunteers ingested controlled diets with a fixed number of calories for 112 d. Samples of breath and urine were collected up to 112 d after administration. Samples of feces were collected up to 14 d after administration. Hair samples were obtained at 119 d after administration and analyzed as a representative index of the rate of excretion of organic 13 C via pathways such as skin cell exfoliation and mucus secretion. All samples were analyzed for 13 C/ 12 C atomic ratio to determine the rate of excretion via each pathway. We then constructed a metabolic model with a total of four pathways (breath, urine, feces, and other) comprising seven compartments. We determined the values of the biokinetic parameters in the model by using the obtained excretion data. From 74% to 94% of the 13 C administered was excreted in breath, whereas    0.1). In addition, the dataset for one of the three subjects was markedly different from those of the other two. When we estimated the 50 year cumulative body burden for 13 C by using our model and we included non-statistically significant parameters, a considerable cumulative body burden was found in the compartments excreting to the other pathway. Although our results on the cumulative body burden of 13 C from orally administered carbon as glucose were inconclusive, we found that the compartments excreting to the other pathway had a markedly long residence time and therefore should be studied further to clarify the fate of carbon in the human body. In addition to excreta, data for serum and blood cell samples were also collected from the subjects to examine the metabolism of 13 C in human body.

  4. Auto-inducing media for uniform isotope labeling of proteins with 15N, 13C and 2H

    International Nuclear Information System (INIS)

    Guthertz, Nicolas; Klopp, Julia; Winterhalter, Aurélie; Fernández, César; Gossert, Alvar D.

    2015-01-01

    Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with 15 N, 13 C and/or 2 H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of 13 C, 15 N of 96.6 % and 2 H, 15 N of 98.8 %. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer

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

    Science.gov (United States)

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

    2005-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Van Dien, Stephen J.

    2005-04-12

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

  7. The cerebral metabolism of amino acids and related metabolites as studied by {sup 13}C and {sup 14}C labelling

    Energy Technology Data Exchange (ETDEWEB)

    Hassel, B

    1995-11-01

    The present investigations show the feasibility of analyzing the cerebral metabolism of amino acids and related metabolites by {sup 13}C-and {sup 14}C-labelling using labelled acetate and glucose as markers for glial and neuronal metabolism, respectively. Using [{sup 13}C]acetate, it was shown that glial cells export {approx}60% of their TCA cycle intermediates, mostly as glutamine, and that this glutamine is used by neurons partly as an energy reserve, and partly it is converted directly to glutamate and GABA. Using [{sup 13}C]glucose, the glial process or pyruvate carboxylation was shown to compensate fully for the loss of glutamine. The mechanism of action of two neurotoxins, fluorocitrate and 3-nitropropionate was elucidated. The latter toxin was shown to inhibit the TCA cycle of GABAergic neurons selectively. Formation of pyruvate and lactate from glial TCA cycle intermediates was demonstrated in vivo. This pathway may be important for glial inactivation of transmitter glutamate and GABA. The results illustrate glianeuronal interactions, and they suggest the applicability of {sup 13}CNMR spectroscopy to the detailed study of the cerebral metabolism of amino acids in the intact, unanesthetized human brain. 174 refs.

  8. The cerebral metabolism of amino acids and related metabolites as studied by 13C and 14C labelling

    International Nuclear Information System (INIS)

    Hassel, B.

    1995-11-01

    The present investigations show the feasibility of analyzing the cerebral metabolism of amino acids and related metabolites by 13 C-and 14 C-labelling using labelled acetate and glucose as markers for glial and neuronal metabolism, respectively. Using [ 13 C[acetate, it was shown that glial cells export ∼60% of their TCA cycle intermediates, mostly as glutamine, and that this glutamine is used by neurons partly as an energy reserve, and partly it is converted directly to glutamate and GABA. Using [ 13 C[glucose, the glial process or pyruvate carboxylation was shown to compensate fully for the loss of glutamine. The mechanism of action of two neurotoxins, fluorocitrate and 3-nitropropionate was elucidated. The latter toxin was shown to inhibit the TCA cycle of GABAergic neurons selectively. Formation of pyruvate and lactate from glial TCA cycle intermediates was demonstrated in vivo. This pathway may be important for glial inactivation of transmitter glutamate and GABA. The results illustrate glianeuronal interactions, and they suggest the applicability of 13 CNMR spectroscopy to the detailed study of the cerebral metabolism of amino acids in the intact, unanesthetized human brain. 174 refs

  9. Experiments and strategies for the assignment of fully13 C/15N-labelled polypeptides by solid state NMR

    International Nuclear Information System (INIS)

    Straus, Suzana K.; Bremi, Tobias; Ernst, Richard R.

    1998-01-01

    High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully 13 C/ 15 N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue 13 C- 15 N correlation experiments with 13 C- 13 C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using 15 N instead of 13 C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the 15 N and 13 C resonances in human ubiquitin

  10. Accurate and sensitive determination of molar fractions of "1"3C-Labeled intracellular metabolites in cell cultures grown in the presence of isotopically-labeled glucose

    International Nuclear Information System (INIS)

    Fernández-Fernández, Mario; Rodríguez-González, Pablo; Hevia Sánchez, David; González-Menéndez, Pedro; Sainz Menéndez, Rosa M.; García Alonso, J. Ignacio

    2017-01-01

    This work describes a methodology based on multiple linear regression and GC-MS for the determination of molar fractions of isotopically-labeled intracellular metabolites in cell cultures. Novel aspects of this work are: i) the calculation of theoretical isotopic distributions of the different isotopologues from an experimentally measured value of % 13C enrichment of the labeled precursor ii) the calculation of the contribution of lack of mass resolution of the mass spectrometer and different fragmentation mechanism such as the loss or gain of hydrogen atoms in the EI source to measure the purity of the selected cluster for each metabolite and iii) the validation of the methodology not only by the analysis of gravimetrically prepared mixtures of isotopologues but also by the comparison of the obtained molar fractions with experimental values obtained by GC-Combustion-IRMS based on "1"3C/"1"2C isotope ratio measurements. The method is able to measure molar fractions for twenty-eight intracellular metabolites derived from glucose metabolism in cell cultures grown in the presence of "1"3C-labeled Glucose. The validation strategies demonstrate a satisfactory accuracy and precision of the proposed procedure. Also, our results show that the minimum value of "1"3C incorporation that can be accurately quantified is significantly influenced by the calculation of the spectral purity of the measured cluster and the number of "1"3C atoms of the labeled precursor. The proposed procedure was able to accurately quantify gravimetrically prepared mixtures of natural and labeled glucose molar fractions of 0.07% and mixtures of natural and labeled glycine at molar fractions down to 0.7%. The method was applied to initial studies of glucose metabolism of different prostate cancer cell lines. - Highlights: • Determination of molar fractions of "1"3C-labeled metabolites in cell cultures. • The method is based on multiple linear regression and GC-MS. • Validation of the method by

  11. Accurate and sensitive determination of molar fractions of {sup 13}C-Labeled intracellular metabolites in cell cultures grown in the presence of isotopically-labeled glucose

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Fernández, Mario [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo (Spain); Rodríguez-González, Pablo, E-mail: rodriguezpablo@uniovi.es [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo (Spain); Hevia Sánchez, David; González-Menéndez, Pedro; Sainz Menéndez, Rosa M. [University Institute of Oncology (IUOPA), University of Oviedo, Julián Clavería 6, 33006 Oviedo (Spain); García Alonso, J. Ignacio [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo (Spain)

    2017-05-29

    This work describes a methodology based on multiple linear regression and GC-MS for the determination of molar fractions of isotopically-labeled intracellular metabolites in cell cultures. Novel aspects of this work are: i) the calculation of theoretical isotopic distributions of the different isotopologues from an experimentally measured value of % 13C enrichment of the labeled precursor ii) the calculation of the contribution of lack of mass resolution of the mass spectrometer and different fragmentation mechanism such as the loss or gain of hydrogen atoms in the EI source to measure the purity of the selected cluster for each metabolite and iii) the validation of the methodology not only by the analysis of gravimetrically prepared mixtures of isotopologues but also by the comparison of the obtained molar fractions with experimental values obtained by GC-Combustion-IRMS based on {sup 13}C/{sup 12}C isotope ratio measurements. The method is able to measure molar fractions for twenty-eight intracellular metabolites derived from glucose metabolism in cell cultures grown in the presence of {sup 13}C-labeled Glucose. The validation strategies demonstrate a satisfactory accuracy and precision of the proposed procedure. Also, our results show that the minimum value of {sup 13}C incorporation that can be accurately quantified is significantly influenced by the calculation of the spectral purity of the measured cluster and the number of {sup 13}C atoms of the labeled precursor. The proposed procedure was able to accurately quantify gravimetrically prepared mixtures of natural and labeled glucose molar fractions of 0.07% and mixtures of natural and labeled glycine at molar fractions down to 0.7%. The method was applied to initial studies of glucose metabolism of different prostate cancer cell lines. - Highlights: • Determination of molar fractions of {sup 13}C-labeled metabolites in cell cultures. • The method is based on multiple linear regression and GC-MS.

  12. Synthesis and biosynthesis of 13C, 15N labeled deoxynucleosides useful for biomolecular structural determinations

    International Nuclear Information System (INIS)

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

    1994-01-01

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

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

    International Nuclear Information System (INIS)

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

    1994-01-01

    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- 13 C, 15 N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2'-deoxy-(amino- 15 N)adenosine (dA). The resulting d(amino- 15 N)A is used in a series of reactions to synthesize 2'-deoxy-(2- 13 C,1,amino- 15 N 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

  14. Methods for sequential resonance assignment in solid, uniformly 13C, 15N labelled peptides: Quantification and application to antamanide

    International Nuclear Information System (INIS)

    Detken, Andreas; Hardy, Edme H.; Ernst, Matthias; Kainosho, Masatsune; Kawakami, Toru; Aimoto, Saburo; Meier, Beat H.

    2001-01-01

    The application of adiabatic polarization-transfer experiments to resonance assignment in solid, uniformly 13 C- 15 N-labelled polypeptides is demonstrated for the cyclic decapeptide antamanide. A homonuclear correlation experiment employing the DREAM sequence for adiabatic dipolar transfer yields a complete assignment of the C α and aliphatic side-chain 13 C resonances to amino acid types. The same information can be obtained from a TOBSY experiment using the recently introduced P9 1 12 TOBSY sequence, which employs the J couplings as a transfer mechanism. A comparison of the two methods is presented. Except for some aromatic phenylalanine resonances, a complete sequence-specific assignment of the 13 C and 15 N resonances in antamanide is achieved by a series of selective or broadband adiabatic triple-resonance experiments. Heteronuclear transfer by adiabatic-passage Hartmann-Hahn cross polarization is combined with adiabatic homonuclear transfer by the DREAM and rotational-resonance tickling sequences into two- and three-dimensional experiments. The performance of these experiments is evaluated quantitatively

  15. Synthesis of 13C-labeled vitamin E and interaction between vitamin E and phospholipid in liposome

    International Nuclear Information System (INIS)

    urano, S.; Matsuo, M.

    1986-01-01

    Vitamin E with a 13 C-labeled isoprenoid side chain, [4'a- 13 C], [6'- 13 C], [8'a- 13 C] and [12'a and 13'- 13 C]α-tocopherols were synthesized. These compounds were incorporated into three kinds of lecithin liposomes from dipalmitoyl phosphatidyl cholin, egg lecithin and rat liver lecithin, of which arachidonic acid contents are 0, 2.6 and 19.0%, respectively. T 1 values, which were measured by NMR for the labeled carbons, indicate that the segmental motion tends to increase with the increase of the distance from the chroman ring. This tendency is not affected with the arachidonic acid contents of phospholipids. This result can not be explained by Lucy's hypothesis. 1 figure; 1 table

  16. Solid state CP/MAS 13C n.m.r. analysis of particle size and density fractions of soil incubated with uniformly labelled 13C-glucose

    International Nuclear Information System (INIS)

    Baldock, J.A.; Oades, J.M.

    1990-01-01

    A soil incubated for 34 days in the absence (control) and presence (treated) of uniformly labelled 13 C-glucose was dispersed using an ultrasonic probe and fractionated by sedimentation in water and a polytungstate solution of density 2.0 Mg m -3 . Solid state CP/MAS 13 C n.m.r. (cross polarization/magic angle spinning 13 C nuclear magnetic resonance) spectroscopy was used to characterize the chemical structure of the native soil organic carbon and the residual substrate carbon in the fractions of the control and treated soils. To obtain quantitative results it was essential to determine the spin lattice relaxation time in a rotating frame of the individual carbon types in the spectra as the relaxation behaviour of the native organic material in the clay fraction was different from that of the residual substrate carbon. The residual substrate carbon was found to accumulate in predominantly alkyl and O-alkyl structures in both fractions. However, significant amounts of acetal and carboxyl carbon were also observed in the clay fraction. Little if any aromatic or phenolic carbon was synthesized by the soil microorganisms utilizing substrate carbon. Dipolar dephasing CP/MAS 13 C n.m.r. experiments were also performed and allowed the proportion of each type of carbon which was protonated and nonprotonated to be estimated. Essentially all of the O-alkyl and acetal carbon, 25-40% of the aromatic carbon and 66-80% of the alkyl carbon was protonated in the fractions isolated from the treated soil. 24 refs., 4 figs., 2 tabs

  17. CACA-TOCSY with alternate {sup 13}C-{sup 12}C labeling: a {sup 13}C{sup {alpha}} direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification

    Energy Technology Data Exchange (ETDEWEB)

    Takeuchi, Koh [National Institute of Advanced Industrial Science and Technology (AIST), Biomedicinal Information Research Center (BIRC) (Japan); Frueh, Dominique P.; Sun, Zhen-Yu J.; Hiller, Sebastian; Wagner, Gerhard, E-mail: gerhard_wagner@hms.harvard.ed [Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology (United States)

    2010-05-15

    We present a {sup 13}C direct detection CACA-TOCSY experiment for samples with alternate {sup 13}C-{sup 12}C labeling. It provides inter-residue correlations between {sup 13}C{sup {alpha}} resonances of residue i and adjacent C{sup {alpha}s} at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C{sup {alpha}} nuclei separated by more than one residue. The experiment also provides C{sup {alpha}}-to-side chain correlations, some amino acid type identifications and estimates for {psi} dihedral angles. The power of the experiment derives from the alternate {sup 13}C-{sup 12}C labeling with [1,3-{sup 13}C] glycerol or [2-{sup 13}C] glycerol, which allows utilizing the small scalar {sup 3}J{sub CC} couplings that are masked by strong {sup 1}J{sub CC} couplings in uniformly {sup 13}C labeled samples.

  18. Selective backbone labelling of ILV methyl labelled proteins

    International Nuclear Information System (INIS)

    Sibille, Nathalie; Hanoulle, Xavier; Bonachera, Fanny; Verdegem, Dries; Landrieu, Isabelle; Wieruszeski, Jean-Michel; Lippens, Guy

    2009-01-01

    Adding the 13 C labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of 12 C labelled glucose instead of the commonly used ( 2 D, 13 C) glucose leads not only to the 13 C labelling of (I, L, V) methyls but also to the selective 13 C labelling of the backbone C α and CO carbons of the Ile and Val residues. As a result, the backbone ( 1 H, 15 N) correlations of the Ile and Val residues and their next neighbours in the (i + 1) position can be selectively identified in HN(CA) and HN(CO) planes. The availability of a selective HSQC spectrum corresponding to the sole amide resonances of the Ile and Val residues allows connecting them to their corresponding methyls by the intra-residue NOE effect, and should therefore be applicable to larger systems

  19. Metabolic fate of 13C-labelled polydextrose and impact on the gut microbiome: A triple-phase study in a colon simulator

    DEFF Research Database (Denmark)

    Lamichhane, Santosh; Yde, Christian C.; Max Jensen, Henrik

    2018-01-01

    The present study introduces a novel triple-phase (liquids, solids and gases) approach, which employed uniformly labelled [U-13C] polydextrose (PDX) for the selective profiling of metabolites generated from dietary fiber fermentation in an in vitro colon simulator using human fecal inocula. Emplo...

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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. (orig.)

  1. Skeleton labeled 13C-carbon nanoparticles for the imaging and quantification in tumor drainage lymph nodes

    Directory of Open Access Journals (Sweden)

    P Xie

    2017-07-01

    Full Text Available Ping Xie,1 Qian Xin,2 Sheng-Tao Yang,3 Tiantian He,2,4 Yuanfang Huang,2 Guangfu Zeng,2,4 Maosheng Ran,2,5 Xiaohai Tang2 1State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, Chengdu, 2Chongqing Lummy Pharmaceutical Co., Ltd, Chongqing, 3College of Chemistry & Environment Protection Engineering, Southwest University for Nationalities, 4College of Life Sciences, Sichuan Normal University, 5State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, People’s Republic of China Abstract: Carbon nanoparticles (CNPs have been widely used in tumor drainage lymph node (TDLN imaging, drug delivery, photothermal therapy, and so on. However, during the theranostic applications, the accumulation efficiency of CNPs in target organs is unknown yet, which largely hinders the extension of CNPs into clinical uses. Herein, we prepared skeleton-labeled 13C-CNPs that had identical properties to commercial CNPs suspension injection (CNSI for the imaging and quantification in TDLN. 13C-CNPs were prepared by arc discharge method, followed by homogenization with polyvinylpyrrolidone. The size distribution and morphology of 13C-CNPs were nearly the same as those of CNSI under transmission electron microscope. The hydrodynamic radii of both 13C-CNPs and CNSI were similar, too. According to X-ray photoelectron spectroscopy and infrared spectroscopy analyses, the chemical compositions and chemical states of elements were also nearly identical for both labeled and commercial forms. The skeleton labeling of 13C was reflected by the shift of G-band toward lower frequency in Raman spectra. 13C-CNPs showed competitive performance in TDLN imaging, where the three lymph nodes (popliteal lymph node, common iliac artery lymph node, and paraaortic lymph node were stained black upon the injection into the hind extremity of mice. The direct quantification of 13C-CNPs indicated that 877 µg

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

    Science.gov (United States)

    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

  3. An inproved synthesis of 24- 13C-labeled bile acids using formyl esters and a modified lead tetraacetate procedure

    International Nuclear Information System (INIS)

    Tserng, K.Y.; Klein, P.D.

    1977-01-01

    An improved synthesis of 24- 13 C-labeled bile acids has been achieved using formyl derivatives of bile acids and a modified lead tetraacetate procedure. The formylated bile acids were degraded by lead tetraacetate and lithium chloride to formylated 23-chloronorcholanes in 72 to 83% yield. Formylated 23-chloronorcholanes were converted to nitriles in dimethylformamide, which were then hydrolyzed to obtain C-24 labeled bile acids in yield of 80 to 90% of labeled sodium cyanide used. This method results in a higher yield and a purer product with less manipulation than previously reported procedures for synthesis of labeled bile acids

  4. One-carbon 13C-labeled synthetic intermediates. Comparison and evaluation of preparative methods

    International Nuclear Information System (INIS)

    Ott, D.G.

    1978-01-01

    Frequently the biggest stumbling block to the synthesis of a structurally complex labeled compound is obtaining the required low molecular weight, structurally simple, isotopic intermediates. Selection of a particular scheme from various alternatives depends on the available capabilities and quantity of product desired, as well as on anticipated future requirements and need for related compounds. Many of the newer reagents for organic synthesis can be applied effectively to isotopic preparations with improvements of yields and simplification of procedures compared to established classical methods. New routes developed for higher molecular weight compounds are sometimes not directly adaptable to the one-carbon analogs, either because of isolation difficulties occasioned by physical properties or by chemical reactivities peculiar to their being first members of homologous series. Various routes for preparation of carbon-13 labeled methanol, formaldehyde, and cyanide are compared

  5. 13C spin relaxation measurements in RNA: Sensitivity and resolution improvement using spin-state selective correlation experiments

    International Nuclear Information System (INIS)

    Boisbouvier, Jerome; Brutscher, Bernhard; Simorre, Jean-Pierre; Marion, Dominique

    1999-01-01

    A set of new NMR pulse sequences has been designed for the measurement of 13 C relaxation rate constants in RNA and DNA bases: the spin-lattice relaxation rate constant R(C z ), the spin-spin relaxation rate constant R(C + ), and the CSA-dipolar cross-correlated relaxation rate constant Γ C,CH xy . The use of spin-state selective correlation techniques provides increased sensitivity and spectral resolution. Sensitivity optimised C-C filters are included in the pulse schemes for the suppression of signals originating from undesired carbon isotopomers. The experiments are applied to a 15% 13 C-labelled 33-mer RNA-theophylline complex. The measured R(C + )/Γ C,CH xy ratios indicate that 13 C CSA tensors do not vary significantly for the same type of carbon (C 2 , C 6 , C 8 ), but that they differ from one type to another. In addition, conformational exchange effects in the RNA bases are detected as a change in the relaxation decay of the narrow 13 C doublet component when varying the spacing of a CPMG pulse train. This new approach allows the detection of small exchange effects with a higher precision compared to conventional techniques

  6. Theoretical Proof and Empirical Confirmation of a Continuous Labeling Method Using Naturally 13C-Depleted Carbon Dioxide

    Institute of Scientific and Technical Information of China (English)

    Weixin Cheng; Feike A. Dijkstra

    2007-01-01

    Continuous isotope labeling and tracing is often needed to study the transformation, movement, and allocation of carbon in plant-soil systems. However, existing labeling methods have numerous limitations. The present study introduces a new continuous labeling method using naturally 13C-depleted CO2. We theoretically proved that a stable level of 13C-CO2 abundance In a labeling chamber can be maintained by controlling the rate of CO2-free air injection and the rate of ambient airflow with coupling of automatic control of CO2 concentration using a CO2 analyzer. The theoretical results were tested and confirmed in a 54 day experiment in a plant growth chamber. This new continuous labeling method avoids the use of radioactive 14C or expensive 13C-enriched CO2 required by existing methods and therefore eliminates issues of radiation safety or unaffordable isotope cost, as well as creating new opportunities for short- or long-term labeling experiments under a controlled environment.

  7. Dynamic nuclear polarization and optimal control spatial-selective 13C MRI and MRS

    DEFF Research Database (Denmark)

    Vinding, Mads Sloth; Laustsen, Christoffer; Maximov, Ivan I.

    2013-01-01

    . This is achieved through the development of spatial-selective single-shot spiral-readout MRI and MRS experiments combined with dynamic nuclear polarization hyperpolarized [1-13C]pyruvate on a 4.7T pre-clinical MR scanner. The method stands out from related techniques by facilitating anatomic shaped region...

  8. [13C] GC-C-IRMS analysis of methylboronic acid derivatives of glucose from liver glycogen after the ingestion of [13C] labeled tracers in rats.

    Science.gov (United States)

    Luengo, Catherine; Azzout-Marniche, Dalila; Fromentin, Claire; Piedcoq, Julien; Lemosquet, Sophie; Tomé, Daniel; Gaudichon, Claire

    2009-11-01

    We developed a complete method to measure low [(13)C] enrichments in glycogen. Fourteen rats were fed a control diet. Six of them also ingested either [U-(13)C] glucose (n=2) or a mixture of 20 [U-(13)C] amino acids (n=4). Hepatic glycogen was extracted, digested to glucose and purified on anion-cation exchange resins. After the optimization of methylboronic acid derivatization using GC-MS, [(13)C] enrichment of extracted glucose was measured by GC-C-IRMS. The accuracy was addressed by measuring the enrichment excess of a calibration curve, which observed values were in good agreement with the expected values (R=0.9979). Corrected delta values were -15.6+/-1.6 delta(13)C (per thousand) for control rats (n=8) and increased to -5 to 8 delta(13)C (per thousand) per thousand and 12-14 delta(13)C (per thousand) per thousand after the ingestion of [U-(13)C] amino acids or [U-(13)C] glucose as oral tracers, respectively. The method enabled the determination of dietary substrate transfer into glycogen. The sequestration of dietary glucose in liver glycogen 4 h after the meal was 35% of the ingested dose whereas the transfer of carbon skeletons from amino acids was only 0.25 to 1%.

  9. Investigations on the isoprenoid biosynthesis in the green alga Scenedesmus obliquus by using the 13C-labelling technique

    International Nuclear Information System (INIS)

    Schwender, J.

    1995-01-01

    The biosynthesis of several prenyllipids (isoprenoid lipids) of the green alga Scendesmus obliquus was investigated. The aim was to verify, whether the biosynthesis of isopentenyl diphosphate (IPP) in Scenedesmus proceeds according to the classical acetate mevalonate pathway or to an alternative pathway. An alternative pathway for IPP formation has recently been detected in some eubacteria by the group of Prof. M. Rohmer. Some inhibition tests were performed with mevinolin, a specific inhibitor of HMG-CoA reductase which yields mevalonic acid. Mevinolin should block the biosynthesis of such isoprenoids which are formed via the acetate mevalonate pathway. Scenedesmus was grown heterotrophically on 13 C-labelled glucose or acetate. After isolation and purification of 13 C-labelled phytol (side chains of chlorophylls), β-carotene, lutein, plastoquinone-9 and three sterol compounds, the enrichment of 13 C at different carbon-positions of the labelled compounds was determined. This was achieved by the 13 C-NMR technique in cooperation with Miriam Seemann of the group of Prof. M. Rohmer in Mullhouse/France. (orig.) [de

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

    Science.gov (United States)

    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.

  11. Affordable uniform isotope labeling with {sup 2}H, {sup 13}C and {sup 15}N in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D., E-mail: alvar.gossert@novartis.com [Novartis Institutes for BioMedical Research (Switzerland)

    2015-06-15

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Koichi; Matsunaga, C.; Igarashi, Takako; Kim, Hahyung; Odaka, Asano; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo, Hongo (Japan))

    1991-01-01

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

  13. Complete assignment of the methionyl carbonyl carbon resonance in switch variant anti-dansyl antibodies labeled with [1-13C]methionine

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  14. CACA-TOCSY with alternate 13C-12C labeling: a 13Cα direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification

    International Nuclear Information System (INIS)

    Takeuchi, Koh; Frueh, Dominique P.; Sun, Zhen-Yu J.; Hiller, Sebastian; Wagner, Gerhard

    2010-01-01

    We present a 13 C direct detection CACA-TOCSY experiment for samples with alternate 13 C- 12 C labeling. It provides inter-residue correlations between 13 C α resonances of residue i and adjacent C α s at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C α nuclei separated by more than one residue. The experiment also provides C α -to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate 13 C- 12 C labeling with [1,3- 13 C] glycerol or [2- 13 C] glycerol, which allows utilizing the small scalar 3 J CC couplings that are masked by strong 1 J CC couplings in uniformly 13 C labeled samples.

  15. CACA-TOCSY with alternate 13C-12C labeling: a 13Calpha direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification.

    Science.gov (United States)

    Takeuchi, Koh; Frueh, Dominique P; Sun, Zhen-Yu J; Hiller, Sebastian; Wagner, Gerhard

    2010-05-01

    We present a (13)C direct detection CACA-TOCSY experiment for samples with alternate (13)C-(12)C labeling. It provides inter-residue correlations between (13)C(alpha) resonances of residue i and adjacent C(alpha)s at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C(alpha) nuclei separated by more than one residue. The experiment also provides C(alpha)-to-sidechain correlations, some amino acid type identifications and estimates for psi dihedral angles. The power of the experiment derives from the alternate (13)C-(12)C labeling with [1,3-(13)C] glycerol or [2-(13)C] glycerol, which allows utilizing the small scalar (3)J(CC) couplings that are masked by strong (1)J(CC) couplings in uniformly (13)C labeled samples.

  16. Chemienzymatic synthesis of Uridine. Nucleotides labeled with [15N] and [13C

    DEFF Research Database (Denmark)

    Gilles, Anne-Marie; Cristea, Ioan; Palibroda, Nicolae

    1995-01-01

    +necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and a-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N,13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended...... and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy....

  17. Retrobiosynthetic NMR studies with 13C-labeled glucose. Formation of gallic acid in plants and fungi

    International Nuclear Information System (INIS)

    Werner, I.; Bacher, A.; Eisenreich, W.

    1997-01-01

    The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this

  18. Correction factors for 13C-labelled substrate oxidation at whole-body and muscle level

    DEFF Research Database (Denmark)

    Van Hall, Gerrit

    1999-01-01

    acid cycle. Changes in metabolic rate induced, for example, by feeding, hormonal changes and physical activity, as well as infusion time, have been shown to affect both correction factors. The present paper explains the theoretical and physiological basis of these correction factors and makes...... for the proportion of labelled CO2 that is produced via oxidation but not excreted. Furthermore, depending on the substrate and position of the C label(s), there may also be a need to correct for labelled C from the metabolized substrate that does not appear as CO2, but rather becomes temporarily fixed in other...

  19. General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

    International Nuclear Information System (INIS)

    Rene, Brigitte; Masliah, Gregoire; Zargarian, Loussine; Mauffret, Olivier; Fermandjian, Serge

    2006-01-01

    Summary 13 C, 15 N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments

  20. Microbial Metabolism in Soil at Subzero Temperatures: Adaptation Mechanisms Revealed by Position-Specific 13C Labeling

    Directory of Open Access Journals (Sweden)

    Ezekiel K. Bore

    2017-05-01

    Full Text Available Although biogeochemical models designed to simulate carbon (C and nitrogen (N dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically 13C-labeled glucose at three temperatures: +5 (control, -5, and -20°C. 13C was quantified in CO2, bulk soil, microbial biomass, and dissolved organic carbon (DOC after 1, 3, and 10 days and also after 30 days for samples at -20°C. Compared to +5°C, CO2 decreased 3- and 10-fold at -5 and -20°C, respectively. High 13C recovery in CO2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5°C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis. A threefold higher 13C recovery in microbial biomass at -5 than +5°C points to synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Less than 0.4% of 13C was recovered in DOC after 1 day, demonstrating complete glucose uptake by microorganisms even at -20°C. Consequently, we attribute the fivefold higher extracellular 13C in soil than in microbial biomass to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. The knowledge of sustained metabolism at subzero temperatures will not only be useful for modeling global C dynamics in ecosystems with periodically or permanently frozen soils, but will also be important in understanding and controlling the adaptive mechanisms of food spoilage organisms.

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

    Science.gov (United States)

    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

  2. Auto-inducing media for uniform isotope labeling of proteins with {sup 15}N, {sup 13}C and {sup 2}H

    Energy Technology Data Exchange (ETDEWEB)

    Guthertz, Nicolas [Institute of Cancer Research, Division of Structural Biology (United Kingdom); Klopp, Julia; Winterhalter, Aurélie; Fernández, César; Gossert, Alvar D., E-mail: alvar.gossert@novartis.com [Novartis Institutes for BioMedical Research (Switzerland)

    2015-06-15

    Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with {sup 15}N, {sup 13}C and/or {sup 2}H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of {sup 13}C, {sup 15}N of 96.6 % and {sup 2}H, {sup 15}N of 98.8 %. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer.

  3. Metabolic Response of Soil Microorganisms to Frost: A New Perspective from Position-specific 13C Labeling

    Science.gov (United States)

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

    2016-12-01

    Cold adapted organisms and their biomolecules have received considerable attention in the last few decades, particularly in light of the perceived biotechnological potential. Mostly, these studies are based on pure isolated cultures from permafrost or permafrost samples with inherently adapted microbes. However, microbial activities in agricultural soils that are predominantly exposed to freeze conditions during winter in temperate ecosystems remain unclear. To analyze microbial metabolism at low soil temperatures, isotopomeres of position-specifically 13C labeled glucose were incubated at three temperature; 5 (control), -5 -20 oC. Soils were sampled after 1, 3 and 10 days (and after 30 days for samples at -20 °C). 13C was quantifed in CO2, bulk soil, microbial biomass and dissolved organic carbon (DOC). Highest 13C recovery in CO2 was obtained from C-1 position in control soil. Consequently, metabolic activity was dominated by pentose phosphate pathway at 5 °C. In contrast, metabolic behaviors switched towards a preferential respiration of the glucose C-4 position at -5 and -20 °C. High 13C recovery from C-4 position confirms previous studies suggesting that fermentation increases at subzero temperature. A 3-fold higher 13C recovery in microbial biomass at -5 °C than under control conditions points towards synthesis of intracellular antifreeze metabolites such as glycerol and ethanol and it is consistent with fermentative metabolism. A 5-fold higher 13C in bulk soil than microbial biomass at -20 °C does not reflect non-metabolized glucose because 13C recovery in DOC was less than 0.4% at day 1. Therefore, high 13C recovery in bulk soil at -20 °C was attributed to extracellular metabolites secreted to overcome frost. The shift in antifreeze mechanisms with temperature was brought about by shift in microbial community structure as indicated by incorporation into 13C into PLFA which was 2-fold higher in gram negative bacteria under control than frozen

  4. Mathematical modeling of (13)C label incorporation of the TCA cycle: the concept of composite precursor function.

    Science.gov (United States)

    Uffmann, Kai; Gruetter, Rolf

    2007-11-15

    A novel approach for the mathematical modeling of (13)C label incorporation into amino acids via the TCA cycle that eliminates the explicit calculation of the labeling of the TCA cycle intermediates is described, resulting in one differential equation per measurable time course of labeled amino acid. The equations demonstrate that both glutamate C4 and C3 labeling depend in a predictable manner on both transmitochondrial exchange rate, V(X), and TCA cycle rate, V(TCA). For example, glutamate C4 labeling alone does not provide any information on either V(X) or V(TCA) but rather a composite "flux". Interestingly, glutamate C3 simultaneously receives label not only from pyruvate C3 but also from glutamate C4, described by composite precursor functions that depend in a probabilistic way on the ratio of V(X) to V(TCA): An initial rate of labeling of glutamate C3 (or C2) being close to zero is indicative of a high V(X)/V(TCA). The derived analytical solution of these equations shows that, when the labeling of the precursor pool pyruvate reaches steady state quickly compared with the turnover rate of the measured amino acids, instantaneous labeling can be assumed for pyruvate. The derived analytical solution has acceptable errors compared with experimental uncertainty, thus obviating precise knowledge on the labeling kinetics of the precursor. In conclusion, a substantial reformulation of the modeling of label flow via the TCA cycle turnover into the amino acids is presented in the current study. This approach allows one to determine metabolic rates by fitting explicit mathematical functions to measured time courses.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-02-01

    Clinically translatable hyperpolarized (HP) (13)C-NMR can probe in vivo enzymatic reactions, e.g., lactate dehydrogenase (LDH)-catalyzed reaction by injecting HP (13)C-pyruvate into the subject, which is converted to (13)C labeled lactate by the enzyme. Parameters such as (13)C-lactate signals and lactate-to-pyruvate signal ratio are commonly used for analyzing the HP (13)C-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 (13)C-NMR data and investigate if they can be potential predictors of lung inflammation. 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 (13)C-pyruvate for injecting into the lungs. A 20 mm (1)H/(13)C dual-tuned coil in a 9.4-T Varian vertical bore NMR spectrometer was employed to acquire the (13)C 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 (13)C labeled pyruvate and lactate. 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 7(th) day post treatment. Wilcoxon rank-sum test showed that the medians of these kinetic parameters of the 7-day cohort were significantly

  7. Synthesis of the mevalonic acid labelled with "1"4C, "1"3C and "3H

    International Nuclear Information System (INIS)

    Rousseau, Bernard

    1982-01-01

    This thesis describes five new methods of synthesis of the (R,S) mevalonic acid adapted to the labelling with "1"4C and "1"3C in positions 4,5 or 5 or 3', or with tritium in position 3'. Three of them use the tri-oxa-2,4,10 adamantyl group as masked carboxyl function. The two others take benefit from the regioselectivity of the bis-hydro-boration of terminal acetylenics by the 9-borabicyclo [3-3-1]nonane. The acylation of the bis-trimethylsilyl lithiomalonate, and the chemistry of dithiannes are also involved. Acetylene and methyl iodide labelled with isotopes are used as cheap base products [fr

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

    Energy Technology Data Exchange (ETDEWEB)

    Ashburn, D.A.; Garcia, K.; Hanners, J.L.; Silks, L.A. III; Unkefer, C.J. [Los Alamos National Laboratory, NM (United States)

    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. NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine.

    Science.gov (United States)

    Pederson, Kari; Chalmers, Gordon R; Gao, Qi; Elnatan, Daniel; Ramelot, Theresa A; Ma, Li-Chung; Montelione, Gaetano T; Kennedy, Michael A; Agard, David A; Prestegard, James H

    2017-07-01

    A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145 kDa dimer. It uses 13 C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from 1 H- 13 C residual dipolar couplings (RDCs) of alanine methyl groups. In all, 40 of the 47 expected crosspeaks were resolved and 36 gave RDC data. Assignments of crosspeaks were partially achieved by transferring assignments from those made on individual domains using triple resonance methods. However, these were incomplete and in many cases the transfer was ambiguous. A genetic algorithm search for consistency between predictions based on domain structures and measurements for chemical shifts and RDCs allowed 60% of the 40 resolved crosspeaks to be assigned with confidence. Chemical shift changes of these crosspeaks on adding an ATP analog to the apo-protein are shown to be consistent with structural changes expected on comparing previous crystal structures for apo- and complex- structures. RDCs collected on the assigned alanine methyl peaks are used to generate a new solution model for the apo-protein structure.

  10. Composition, production, and loss of carbohydrates in subtropical shallow subtidal sandy sediments: Rapid processing and long-term retention revealed by 13C-labeling

    NARCIS (Netherlands)

    Oakes, J.M.; Eyre, B.D.; Middelburg, J.J.; Boschker, H.T.S.

    2010-01-01

    The composition and production of carbohydrates (mannose, rhamnose, fucose, galactose, glucose, and xylose) and their transfer among sediment compartments (microphytobenthos [MPB], bacteria, and detritus) was investigated through in situ labeling with 13C-bicarbonate. After 60 h, 13C was found in

  11. "VALIDATION OF 13C-UREA BREATH TEST WITH NON DISPERSIVE ISOTOPE SELECTIVE INFRARED SPECTROSCOPY FOR THE DIAGNOSIS OF HELICOBACTER PYLORI INFECTION: A SURVEY IN IRANIAN POPULATION"

    Directory of Open Access Journals (Sweden)

    "Davood Beiki

    2005-04-01

    Full Text Available The urea breath test (UBT which is carried out with 13C or 14C labeled urea is one of the most important non invasive methods for detection of Helicobacter pylori infection. Application of 13C-UBT is becoming increasingly popular because of its non radioactive nature which makes it suitable for diagnostic purposes in children and women of child bearing ages. While isotope ratio mass spectrometer (IRMS is generally used to detect 13C in expired breath, this instrument is expensive and recently non dispersive isotope selective infrared (NDIR spectroscopy which is a lower cost technique has been employed as a reliable counterpart for IRMS in small clinics. The aim of this study was to assess the validity of NDIR spectroscopy technique in Iranian population in comparison with histological examination, rapid urease test and 14C-urea breath test as gold standard. Seventy six patients with dyspepsia were underwent 13CUBT for diagnosis of Helicobacter pylori infection. Good agreements were found between the 13C-UBT and gold standard methods. The 13C-UBT showed 100% sensitivity, 97.3% specificity, 97.56% positive predictive value, 100% negative predictive value and 98.65% accuracy. On the basis of these results it could be concluded that 13C-UBT performed with NDIR spectroscopy is a reliable, accurate and non invasive diagnostic tool for detection of Helicobacter pylori infection in the Iranian population.

  12. The transformation and fate of sub-Arctic microphytobenthos carbon revealed through 13C-labeling

    DEFF Research Database (Denmark)

    Oakes, Joanne M.; Rysgaard, Søren; Glud, Ronnie N.

    2016-01-01

    Microphytobenthos (MPB) at higher latitudes has been poorly studied. This study used pulse-chase C-13-labeling to investigate the production, processing, and fate of MPB-derived carbon (MPB-C) in sub-Arctic intertidal sediments over 31 d. Gross primary production (2.1 mmolCm(-2)h(-1)+/- 0.4 mmol...... accounted for49.8% of this C-13. The C-13 content of sediment organic carbon declined over time, but>31% of the C-13 fixed within the first tidal cycle remained after 31 d, suggesting that sub-Arctic MPB may contribute to coastal carbon retention during the productive season. Over 21 d, 10.6% of the fixed C......-13 was removed via DIC fluxes and 0.3% via DOC fluxes from inundated sediment, and 0.6% as CO2 from exposed sediment. The greatest loss of C-13 (38.2%) was via unmeasured pathways, including resuspension and/or removal by mobile consumers. The rates of MPB-C production and the relative importance...

  13. Nuclear magnetic resonance study of interaction of ligands with Streptococcus faecium dihydrofolate reductase labeled with [#betta#-13C]tryptophan

    International Nuclear Information System (INIS)

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

    1982-01-01

    Dihydrofolate reductase from Streptococcus faecium has been labeled with [#betta#- 13 C]tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the 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 #betta#/ 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

  14. Interpretation of non-invasive breath tests using 13C-labeled substrates - a preliminary report with 13C-methacetin

    Directory of Open Access Journals (Sweden)

    Lock JF

    2009-12-01

    Full Text Available Abstract Non-invasive breath tests can serve as valuable diagnostic tools in medicine as they can determine particular enzymatic and metabolic functions in vivo. However, methodological pitfalls have limited the actual clinical application of those tests till today. A major challenge of non-invasive breath tests has remained the provision of individually reliable test results. To overcome these limitations, a better understanding of breath kinetics during non-invasive breaths tests is essential. This analysis compares the breath recovery of a 13C-methacetin breath test with the actual serum kinetics of the substrate. It is shown, that breath and serum kinetics of the same test are significantly different over a period of 60 minutes. The recovery of the tracer 13CO2 in breath seems to be significantly delayed due to intermediate storage in the bicarbonate pool. This has to be taken into account for the application of non-invasive breath test protocols. Otherwise, breath tests might display bicarbonate kinetics despite the metabolic capacity of the particular target enzyme.

  15. Sparse "1"3C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins

    International Nuclear Information System (INIS)

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

    2016-01-01

    We demonstrate a novel sparse "1"3C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically "1"3C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

  16. Use of 13NMR to delineate the mode of association or binding of 13C-labeled pollutants with humic materials

    International Nuclear Information System (INIS)

    Bortiatynski, J.M.; Minard, R.D.; Hatcher, P.G.

    1993-01-01

    13 C NMR has recently been shown to be a powerful technique for the examination of the covalent binding of pollutants to humic materials when the latter are enriched with 13 C. Enhanced signals are observed for the carbons that are highly enriched with 13 C while the remaining signals due to naturally abundant 13 C form unlabeled pollutant carbons or humic substances are at the baseline noise level. If covalent bonding and/or non covalent associations take place at or near the site of the 13 C label(s), the nature of the bonding or association can be discerned and the adsorption coefficients can be calculated. In this paper, the authors present the results of such binding studies which demonstrate the great potential of this technique

  17. Sampling technique for collection of expired COsub(2) in studies using naturally labelled sup(13)C in calves

    International Nuclear Information System (INIS)

    Chevalier, R.; Pelletier, G.; Gagnon, M.

    1984-01-01

    Natural isotopic labelling of animal metabolic processes presents great advantages over the use of artificially enriched compounds. These advantages include the use of natural diets, low cost and easiness of handling, sampling and disposal. Changes in the sup(13)C/sup(12)C ratio of expired COsub(2) after ingestion of a test meal containing a variable proportion of Csub(3) and/or Csub(4) plant-derived products permits measurements of the immediate oxidation of dietary carbohydrates. A breathing device for collection of expired COsub(2) and purification techniques are described. The breath-sampling device is composed of a polypropylene enclosure and a rubber bag. The rubber bag is blown up by the animal and a sample of the expired gas is transferred to a sampling glass holder in which a vacuum had been previously achieved. Sample purification of expired COsub(2) is done by differential freezing

  18. Identification of an Epoxide Metabolite of Lycopene in Human Plasma Using 13C-Labeling and QTOF-MS

    Directory of Open Access Journals (Sweden)

    Morgan J. Cichon

    2018-03-01

    Full Text Available The carotenoid lycopene is a bioactive component of tomatoes and is hypothesized to reduce risk of several chronic diseases, such as prostate cancer. The metabolism of lycopene is only beginning to be understood and some studies suggest that metabolites of lycopene may be partially responsible for bioactivity associated with the parent compound. The detection and characterization of these compounds in vivo is an important step in understanding lycopene bioactivity. The metabolism of lycopene likely involves both chemical and enzymatic oxidation. While numerous lycopene metabolites have been proposed, few have actually been identified in vivo following lycopene intake. Here, LC-QTOF-MS was used along with 13C-labeling to investigate the post-prandial oxidative metabolism of lycopene in human plasma. Previously reported aldehyde cleavage products were not detected, but a lycopene 1,2-epoxide was identified as a new candidate oxidative metabolite.

  19. Identification of an Epoxide Metabolite of Lycopene in Human Plasma Using 13C-Labeling and QTOF-MS.

    Science.gov (United States)

    Cichon, Morgan J; Moran, Nancy E; Riedl, Ken M; Schwartz, Steven J; Clinton, Steven K

    2018-03-20

    The carotenoid lycopene is a bioactive component of tomatoes and is hypothesized to reduce risk of several chronic diseases, such as prostate cancer. The metabolism of lycopene is only beginning to be understood and some studies suggest that metabolites of lycopene may be partially responsible for bioactivity associated with the parent compound. The detection and characterization of these compounds in vivo is an important step in understanding lycopene bioactivity. The metabolism of lycopene likely involves both chemical and enzymatic oxidation. While numerous lycopene metabolites have been proposed, few have actually been identified in vivo following lycopene intake. Here, LC-QTOF-MS was used along with 13 C-labeling to investigate the post-prandial oxidative metabolism of lycopene in human plasma. Previously reported aldehyde cleavage products were not detected, but a lycopene 1,2-epoxide was identified as a new candidate oxidative metabolite.

  20. 13C-TRIPLY Labeled Ethyl Cyanide Submillimeterwave Study with Lille's Fast Scan Dds-Based Spectrometer

    Science.gov (United States)

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

    2016-06-01

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

  1. Screening and selection of high carotenoid producing in vitro tomato cell culture lines for [13C]-carotenoid production.

    Science.gov (United States)

    Engelmann, Nancy J; Campbell, Jessica K; Rogers, Randy B; Rupassara, S Indumathie; Garlick, Peter J; Lila, Mary Ann; Erdman, John W

    2010-09-22

    Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-labeling. Different Solanum lycopersicum allelic variants for high lycopene and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture, and cell suspension cultures of the hp-1 line were chosen for isotopic labeling. When grown with [U]-13C-glucose and treated with CPTA, hp-1 suspensions yielded highly enriched 13C-lycopene with 45% of lycopene in the M+40 form and 88% in the M+35 to M+40 isotopomer range. To the authors' knowledge this is the first report of highly enriched 13C-carotenoid production from in vitro plant cell culture.

  2. More than a century of Grain for Green Program is expected to restore soil carbon stock on alpine grassland revealed by field {sup 13}C pulse labeling

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qi; Chen, Dongdong; Zhao, Liang [Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai (China); Yang, Xue [Department of Education of Qinghai Province, Xining 810008, Qinghai (China); Xu, Shixiao [Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai (China); Zhao, Xinquan, E-mail: xqzhao@nwipb.cas.cn [Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai (China); Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 10041, Sichuan (China)

    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 ~ 50 years. As a result, the Grain for Green Program (GfGP) has been widely implemented over the last 10 years 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 {sup 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 CO{sub 2} uptake rates in the GfGP grassland than that in the natural grassland. Partitioning of photoassimilate (% of recovered {sup 13}C) in C pools of both grasslands was similar 25 days after labeling, except in the roots of the 0–15 and 5–15 cm soil layer. Soil organic C (SOC) sequestration rate in the GfGP grassland was 11.59 ± 1.89 g C m{sup −2} yr{sup −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 ± 20.26 yr) 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. - Highlights: • Grain for Green Project initiated in 1999 converts cropland to grassland/shrubland. • Impact of Grain for Green on carbon cycling on Qinghai–Tibetan Plateau is unknown. • Effects on carbon partitioning and turnover were accessed by {sup 13}CO{sub 2} pulse labeling. • Different mass of {sup 13}C in excess, similar {sup 13}C partitioning are shown in grasslands. • Soil organic carbon of

  3. More than a century of Grain for Green Program is expected to restore soil carbon stock on alpine grassland revealed by field "1"3C pulse labeling

    International Nuclear Information System (INIS)

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

    2016-01-01

    Anthropogenic changes in land use/cover have altered the vegetation, soil, and carbon (C) cycling on the Qinghai–Tibetan Plateau (QTP) over the last ~ 50 years. As a result, the Grain for Green Program (GfGP) has been widely implemented over the last 10 years 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 "1"3C 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 CO_2 uptake rates in the GfGP grassland than that in the natural grassland. Partitioning of photoassimilate (% of recovered "1"3C) in C pools of both grasslands was similar 25 days after labeling, except in the roots of the 0–15 and 5–15 cm soil layer. Soil organic C (SOC) sequestration rate in the GfGP grassland was 11.59 ± 1.89 g C m"−"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 ± 20.26 yr) 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. - Highlights: • Grain for Green Project initiated in 1999 converts cropland to grassland/shrubland. • Impact of Grain for Green on carbon cycling on Qinghai–Tibetan Plateau is unknown. • Effects on carbon partitioning and turnover were accessed by "1"3CO_2 pulse labeling. • Different mass of "1"3C in excess, similar "1"3C partitioning are shown in grasslands. • Soil organic carbon of cropland will be restored to natural

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

    Energy Technology Data Exchange (ETDEWEB)

    Strzelecka, T.; Gronenborn, A.M.; Clore, G.M. [National Institutes of Health, Bethesda, MD (United States)

    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.

  5. Medium-term response of microbial community to rhizodeposits of white clover and ryegrass and tracing of active processes induced by 13C and 15N labelled exudates

    DEFF Research Database (Denmark)

    Kusliene, Gedrime; Rasmussen, Jim; Kuzyakov, Yakov

    2014-01-01

    and actinomycetes was unaffected by plant species, but pool of Gram-negative and Gram-positive bacteria was greater under white clover at the 10 percent significance level. In the short term, microorganisms more actively utilised fresh exudates (13C-labelled) of ryegrass than of white clover. We expected ryegrass...... microbial groups in soil under white clover (Trifolium repens L.) and ryegrass (Lolium perenne L.) following leaf-labelling with 13C-bicarbonate and 15N-urea. In this way microbial N and 15N and the composition of PLFAs reflect the medium-term (two months) response of microorganisms to rhizodeposits......, whereas the 13C-label of the PLFAs reflects the short-term (one week) utilisation of root exudates following labelling of shoots. In the medium term, microbial biomass N and 15N were greater under the ryegrass, whereas total PLFA was higher under white clover. The relative abundance of fungi...

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

    Directory of Open Access Journals (Sweden)

    A. de Kluijver

    2013-03-01

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

  7. Screening and Selection of High Carotenoid Producing in Vitro Tomato Cell Culture Lines for [13C]-Carotenoid Production

    OpenAIRE

    Engelmann, Nancy J.; Campbell, Jessica K.; Rogers, Randy B.; Rupassara, S. Indumathie; Garlick, Peter J.; Lila, Mary Ann; Erdman, John W.

    2010-01-01

    Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)-triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-lab...

  8. Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry

    International Nuclear Information System (INIS)

    Inbar, L.; Lapidot, A.

    1988-01-01

    Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U- 13 C]fructose and 13 C- and 15 N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamte source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13 C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13 C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose

  9. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Armelle Cabin-Flaman

    2016-06-01

    Full Text Available 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 13C14N- 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.

  10. Studies on /sup 13/C-NMR spectroscopy of carbohydrates. Application of selective deuteration

    Energy Technology Data Exchange (ETDEWEB)

    Mizutani, Kenji; Kajita, Hitomi; Tashima, Takako; Tanaka, Osamu (Hiroshima Univ. (Japan). School of Medicine)

    1982-10-01

    Recently, Stuart et al. have reported that on heating with deuterated Raney nickel in D/sub 2/O, hexopyranosides and oligosaccharides were deuterated selectively at carbon atoms bound to a free hydroxyl group. For the application of this procedure to chemistry and biochemistry of more complex carbohydrates, further study on this reaction was conducted. On prolonged treatment with Raney nickel (24h), methyl ..cap alpha..-L-arabinopyranoside (4) and ..beta..-D-xylopyranoside (5) were partially epimerized to give (5) and methyl ..cap alpha..-L-lyxopyranoside (6) from (4), and methyl ..beta..-D-ribopyranoside (7) and (4) from (5), respectively. Methyl ..cap alpha..-L-rhamnopyranoside (8) was epimerized more slowly to yield methyl 6-deoxy-..cap alpha..-L-glucopyranoside (9). The best conditions for the deuteration under minimizing the epimerization was refluxing with W-7 type deuterated Raney nickel in D/sub 2/O-dioxane (1 : 1) or methanol-d/sub 4/-D/sub 2/O (1 : 1) for about 9h. Methyl ..beta..-sophoroside (13), methyl ..beta..-cellobioside (23) and ..beta..-sophorosyl and ..beta..-gentiobiosyl units of ginsenoside-Rb/sub 1/ (24), a Ginseng-saponin, were deuterated as expected under these conditions, while C-2 and 4 of the inner ..beta..-glucopyranosyl moiety of methyl ..beta..-laminaribioside (18) and C-4 of the inner ..beta..-glucopyranosyl moiety of methyl 2,3-di-O-..beta..-D-glucopyranosyl-..beta..-D-glucopyranoside (22) remained almost undeuterated. Deuteration of ..cap alpha..-1,6-glucan (25) proceeded rather slowly than those of mono- and oligosaccharide to obtain the expected deuteration after treatment for 24h. In the case of the deuteration of cis- and dl-trans-1, 2-cyclohexanediols (11) and ( (12), both deuteration and epimerization proceeded very fast to give an equilibrated mixture of (11-d/sub 2/) and (12-d/sub 2/) within 9h, while the isolated carbinyl carbon (C-12) of (24) was not deuterated. Further, by means of the above selective deuteration, /sup 13

  11. On structural identifiability analysis of the cascaded linear dynamic systems in isotopically non-stationary 13C labelling experiments.

    Science.gov (United States)

    Lin, Weilu; Wang, Zejian; Huang, Mingzhi; Zhuang, Yingping; Zhang, Siliang

    2018-06-01

    The isotopically non-stationary 13C labelling experiments, as an emerging experimental technique, can estimate the intracellular fluxes of the cell culture under an isotopic transient period. However, to the best of our knowledge, the issue of the structural identifiability analysis of non-stationary isotope experiments is not well addressed in the literature. In this work, the local structural identifiability analysis for non-stationary cumomer balance equations is conducted based on the Taylor series approach. The numerical rank of the Jacobian matrices of the finite extended time derivatives of the measured fractions with respect to the free parameters is taken as the criterion. It turns out that only one single time point is necessary to achieve the structural identifiability analysis of the cascaded linear dynamic system of non-stationary isotope experiments. The equivalence between the local structural identifiability of the cascaded linear dynamic systems and the local optimum condition of the nonlinear least squares problem is elucidated in the work. Optimal measurements sets can then be determined for the metabolic network. Two simulated metabolic networks are adopted to demonstrate the utility of the proposed method. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Bacterial production of site specific {sup 13}C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L., E-mail: robert.mcfeeters@uah.edu [University of Alabama in Huntsville, Department of Chemistry (United States)

    2017-01-15

    Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific {sup 13}C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct {sup 13}C chemical shifts and multiple magnetically equivalent {sup 1}H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with {sup 13}C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated {sup 1}H-{sup 13}C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

  13. HPLC-MS Determination of Acrolein and Acetone Generated from 13C3 -Labeled Glycerol Added to Cigarette Tobacco Using Two Machine-Smoking Regimes

    Directory of Open Access Journals (Sweden)

    Yip SH

    2014-12-01

    Full Text Available The extent of blend glycerol degradation in a burning cigarette to form acrolein and acetone has been quantitatively determined by the addition of glycerol-13C3 to three styles of a leading commercial cigarette brand. Multiple Cambridge pads soaked with a solution of 2,4-dinitrophenylhydrazine (DNPH were employed to trap hydrazone derivatives of low molecular weight carbonyl compounds in both mainstream and sidestream smoke. High performance liquid chromatography coupled with negative ion mass spectrometry was used to isolate DNPH derivatives of the volatile carbonyl products of combustion and to ascertain their concentration. Acrolein, acetone, and propionaldehyde were the principal compounds of interest. The DNPH derivatives of acrolein-13C3 and acetone-13C3 were independently synthesized, and they served as external standards for absolute quantitation. The cost of fully labeled propionaldehyde precluded its use in this study. The brand styles selected for study represent the cigarette design features that are most prevalent in the U.S. market today and afford a representative range of standardized “tar” yields (14, 10, and 5 mg/cig, respectively by the Cambridge Filter Method. The brand styles studied are part of a commercial cigarette brand family that does not contain additives to the tobacco blend, including glycerol. Mainstream smoke was generated by an automated smoking machine employing the standard Cambridge Filter Smoking Regime and a more intense regime requiring larger, more frequent puffs and 100% vent blocking that is specified for regulatory purposes by the Canadian federal government. The research indicated that only a small fraction of added glycerol (~0.25%-0.30%, w/w was converted to the two compounds of interest, with the larger portion generally observed in sidestream smoke. Less than 0.1% of the added glycerol was converted to acrolein in mainstream smoke for all cigarette designs and smoking regimes studied.

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

    Energy Technology Data Exchange (ETDEWEB)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan, E-mail: Stephan.Grzesiek@unibas.ch [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland)

    2015-07-15

    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 {sup 15}N and {sup 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.

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

    International Nuclear Information System (INIS)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-01-01

    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

  16. Investigating CH4 production in an oxic plant-soil system -a new approach combining isotopic labelling (13C) and inhibitors

    Science.gov (United States)

    Lenhart, Katharina; Keppler, Frank

    2017-04-01

    Typically, aerated soil are net sinks of atmospheric methane (CH4), being highest in native ecosystems (pristine forests > managed forests > grasslands > crop fields). However, this does not exclude a simultaneous endogenic CH4 production in the plant-soil system, which cannot be detected simply via CH4 flux measurements. Methanogenic archaea producing CH4 under anoxic conditions were thought to be the only biotic source of CH4 in the soil. However, until recently a non-archaeal pathway of CH4 formation is known where CH4 is produced under oxic conditions in plants (Keppler et al. 2006) and fungi (Lenhart et al. 2012). Additionally, abiotic formation of CH4 from soil organic matter was reported (Jugold et al. 2012) and may be ubiquitous in terrestrial ecosystems. The major goal of this project was to determine soil endogenic CH4 sources and to estimate their contribution to the endogenic CH4 production. Especially the effect of plants and fungi on soil CH4 production was investigated. Therefore, a series of experiments was carried out on field fresh soil collected in a grassland and a forest ecosystem under controlled laboratory conditions. By combining selective inhibitors and 13C labelling, CH4 production rates of several CH4 sources were quantified. The major difficulty was to detect the comparatively small flux of CH4 production against the background of the high CH4 consumption rates due to methanotrophic bacteria. Therefore, we supplemented bare soil and soil with vegetation with selective inhibitors and 13C labelled substrates in a closed chamber system. In a first step, CH4 production was determined by the inhibition of CH4 oxidizing bacteria with Difluoromethane (DFM, 2ml l-1). In the following, a 13C labelled substrate (either CO2, Acetate, or Methionine -S-CH3 labelled) was added in combination with a specific inhibitor -either for archaeal methanogenesis (Bromoethanesulfonate), bacteria (Streptomycin), or fungi (Captan, Cycloheximide). Gas samples were

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

    Science.gov (United States)

    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.

  18. Identification of risk factors for pancreatic exocrine insufficiency after pancreaticoduodenectomy using a 13C-labeled mixed triglyceride breath test.

    Science.gov (United States)

    Hirono, Seiko; Murakami, Yoshiaki; Tani, Masaji; Kawai, Manabu; Okada, Ken-ichi; Uemura, Kenichiro; Sudo, Takeshi; Hashimoto, Yasushi; Nakagawa, Naoya; Kondo, Naru; Yamaue, Hiroki

    2015-02-01

    There are only a few reports concerning long-term exocrine function after pancreaticoduodenectomy (PD), although the number of long-term survivors has increased. We assessed pancreatic exocrine function after PD in 189 patients to identify risk factors for pancreatic exocrine insufficiency. We evaluated patients' exocrine function by using the (13)C-labeled mixed triglyceride breath test, a noninvasive test feasible in outpatient service units. The present study included 99 patients that underwent pancreaticojejunostomy (PJ) at Wakayama Medical University Hospital and 90 patients that underwent pancreaticogastrostomy (PG) at Hiroshima University Hospital, the standard reconstruction techniques during PD at the respective hospitals. We also analyzed long-term morphological changes of remnant pancreas by computed tomography (main pancreatic duct dilation and parenchymal atrophy), nutritional status, and endocrine function. Independent risk factors for exocrine insufficiency after PD include hard pancreas (P = 0.003, odds ratio; 3.157) and PG reconstruction (P = 0.040, odds ratio; 2.321). Breath test results correlated significantly with post-operative morphological changes, nutritional status, and endocrine function. Atrophic changes of the remnant pancreas in the PG group were more severe than those in the PJ group. Furthermore, for patients with a soft pancreas, postoperative body weight changes, prognostic nutritional index, serum total protein levels as well as exocrine test were worse in the PG group, compared with the PJ group. Our results showed that PJ reconstruction might be superior to PG during PD, from the viewpoint of long-term pancreatic exocrine function, although further prospective studies are needed.

  19. Studies of the intermediary metabolism in cultured cells of the insect Spodoptera frugiperda using 13C- or 15N-labelled tracers

    Directory of Open Access Journals (Sweden)

    Bacher Adelbert

    2005-11-01

    Full Text Available Abstract Background Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of 15N/13C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-13C6]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of 15N-phenylalanine. Results Quantitative NMR analysis showed incorporation of the proffered [U-13C6]glucose into the ribose moiety of ribonucleosides (40 – 45% and into the amino acids, alanine (41%, glutamic acid/glutamine (C-4 and C-5, 30% and aspartate/asparagine (15%. Other amino acids and the purine ring of nucleosides were not formed from exogenous glucose in significant amounts (> 5%. Prior to the incorporation into protein the proffered 15N-phenylalanine lost about 70% of its label by transamination and the labelled compound was not converted into tyrosine to a significant extent. Conclusion Growth of S. frugiperda cells in the presence of [U-13C6]glucose is conducive to the fractional labelling of ribonucleosides, alanine, glutamic acid/glutamine and aspartic acid/asparagine. The isotopolog compositions of the ribonucleosides and of alanine indicate considerable recycling of carbohydrate intermediates in the reductive branch of the pentose phosphate pathway. The incorporation of 15N-labelled amino acids may be hampered by loss of the 15N-label by transamination.

  20. Synthesis of {sup 13}C- and {sup 14}C-labeled 1192U90, an ortho-amino benzamide with a preclinical atypical antipsychotic profile

    Energy Technology Data Exchange (ETDEWEB)

    Norman, M.H.; Gabriel, S.D. [Glaxo Wellcome Inc., Research Triangle Park, NC (United States)

    1996-03-01

    Three isotopic forms of potential antipsychotic agent 1192U90 (2-amino-N-(4-(4-(1,2-benzisthiazol-3-yl)-piperazinyl)butyl)benzam ide) were synthesized: one containing {sup 13}C-isotopes and two containing {sup 14}C-isotopes. The compound in which the ortho-amino benzamide ring is completely {sup 13}C-labeled was prepared in a four-step sequence starting from [{sup 13}C{sub 6}]aniline. The {sup 14}C-labeled compounds were prepared by methods analogous to those previously described for the unlabeled material. The key step involved the condensation of 3-(4-(4aminobutyl)-1-piperazinyl)-1,2-benzisothiazole with isatoic anhydride. The first {sup 14}C-labeled compound (3) was prepared from {sup 14}C-labeled 3-(4-(4-aminobutyl)-1-piperazinyl)-1,2-benzisothiazole, while the second compound (4) derived its isotopic label from [{sup 14}C]isatoic anhydride. Compound 3 had a specific activity of 26.55 mCi/mmol, a radiochemical purity of 99.3%, and a radiochemical yield of 3.4%. Compound 4 had a specific activity of 22.67 mCi/mmol and a radiochemical purity of 99.2%. (author).

  1. 13C- and 15N-labelled non-biogenic compounds used as stable isotope drugs for human liver function tests

    International Nuclear Information System (INIS)

    Krumbiegel, P.

    1989-01-01

    As a result of liver diseases, the elimination of certain drugs is retarded. After labelling a suitable drug with 13 C, the 13 CO 2 elimination rate serves as a liver function parameter. Current contributions to the 13 CO 2 breath test method are reviewed and related to the 14 CO 2 breath test proposals. In spite of several advantages of 13 C-labelled agents, some dissatisfaction has remained with the tests, especially at using them with infants. It is the necessity of face masks and the uncertainty to consider endogeneous CO 2 contributions diluting the exhaled 13 CO 2 . The problems are avoided if the other molecule site of the drug is labelled which is known to be eliminated via urine. With 15 N as a tracer, a suitable urine test using [ 15 N]-methacetin as agent has been proposed and put into practice. (author)

  2. Carbon isotope discrimination during litter decomposition can be explained by selective use of substrate with differing δ13C

    Science.gov (United States)

    Ngao, J.; Cotrufo, M. F.

    2011-01-01

    Temporal dynamics of C isotopic composition (δ13C) of CO2 and leaf litter was monitored during a litter decomposition experiment using Arbutus unedo L., as a slow decomposing model substrate. This allowed us (1) to quantify isotopic discrimination variation during litter decomposition, and (2) to test whether selective substrate use or kinetic fractionation could explain the observed isotopic discrimination. Total cumulative CO2-C loss (CL) comprised 27% of initial litter C. Temporal evolution of CL was simulated following a three-C-pool model. Isotopic composition of respired CO2 (δRL) was higher with respect to that of the bulk litter. The isotopic discrimination Δ(L/R) varied from -2‰ to 0‰ and it is mostly attributed to the variations of δRL. A three-pool model, with the three pools differing in their δ13C, described well the dynamic of Δ(L/R), in the intermediate stage of the process. This suggests that the observed isotopic discrimination between respired CO2 and bulk litter is in good agreement with the hypothesis of successive consumption of C compounds differing in δ13C during decomposition. However, to explain also 13C-CO2 dynamics at the beginning and end of the incubation the model had to be modified, with discrimination factors ranging from -1‰ to -4.6‰ attributed to the labile and the recalcitrance pool, respectively. We propose that this discrimination is also the result of further selective use of specific substrates within the two pools, likely being both the labile and recalcitrant pool of composite nature. In fact, the 2‰ 13C enrichment of the α-cellulose observed by the end of the experiment, and potentially attributable to kinetic fractionation, could not explain the measured Δ(L/R) dynamics.

  3. The fate of 13C15N labelled glycine in permafrost and surface soil at simulated thaw in mesocosms from high arctic and subarctic ecosystems

    DEFF Research Database (Denmark)

    Ravn, Nynne Marie Rand; Elberling, Bo; Michelsen, Anders

    2017-01-01

    Background and aim: Nutrient distribution and carbon fluxes upon spring thaw are compared in mesocosms from high arctic and subarctic ecosystems dominated by Cassiope tetragona or Salix hastata/Salix arctica, in order to evaluate the possibility of plant and microbial utilization of an organic...... compound in thawing permafrost and surface soil. Methods: Double labeled glycine (13C15N) was added to soil columns with vegetation and to permafrost. During thaw conditions ecosystem respiration 13C was measured and 13C and 15N distribution in the ecosystem pools was quantified one day and one month after...... glycine addition. Results: Near-surface soil microbes were more efficient in the uptake of intact glycine immediately upon thaw than plants. After one month plants had gained more 15N whereas microbes seemed to lose 15N originating from glycine. We observed a time lag in glycine degradation upon...

  4. Transformation and fate of microphytobenthos carbon in subtropical shallow subtidal sands: A 13C-labeling study

    NARCIS (Netherlands)

    Oakes, J.M.; Eyre, B.D.; Middelburg, J.J.

    2012-01-01

    Microphytobenthos (MPB) in photic sediments are highly productive but the fate of this production remains uncertain. Over 33 d, tracing of 13C from added bicarbonate in subtropical shallow subtidal sand showed rapid transfer of MPB-derived carbon to deeper sediment; below 2 cm (31% within 60 h)

  5. Evidence for in situ degradation of mono-and polyaromatic hydrocarbons in alluvial sediments based on microcosm experiments with 13C-labeled contaminants

    International Nuclear Information System (INIS)

    Morasch, B.; Hoehener, P.; Hunkeler, D.

    2007-01-01

    A microcosm study was conducted to investigate the degradation of mono- and polyaromatic hydrocarbons under in situ-like conditions using alluvial sediments from the site of a former cokery. Benzene, naphthalene, or acenaphthene were added to the sediments as 13 C-labeled substrates. Based on the evolution of 13 C-CO 2 determined by gas chromatography isotope-ratio mass spectrometry (GC-IRMS) it was possible to prove mineralization of the compound of interest in the presence of other unknown organic substances of the sediment material. This new approach was suitable to give evidence for the intrinsic biodegradation of benzene, naphthalene, and acenaphthene under oxic and also under anoxic conditions, due to the high sensitivity and reproducibility of 13 C/ 12 C stable isotope analysis. This semi-quantitative method can be used to screen for biodegradation of any slowly degrading, strongly sorbing compound in long-term experiments. - A method based on 13 C-labeled substrates was developed to determine the intrinsic biodegradation potential of aromatic pollutants under oxic and under anoxic conditions

  6. Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly 13C-Labeled Standards Derived from Spirulina.

    Science.gov (United States)

    Schatschneider, Sarah; Abdelrazig, Salah; Safo, Laudina; Henstra, Anne M; Millat, Thomas; Kim, Dong-Hyun; Winzer, Klaus; Minton, Nigel P; Barrett, David A

    2018-04-03

    We have investigated the applicability of commercially available lyophilized spirulina ( Arthrospira platensis), a microorganism uniformly labeled with 13 C, as a readily accessible source of multiple 13 C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)- 13 C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze-thaw, homogenization, and sonication methods. The limits of quantification were ≤1 μM with excellent linearity for all of the calibration curves ( R 2 ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood-Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U- 13 C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation.

  7. A Comparison between Radiolabeled Fluorodeoxyglucose Uptake and Hyperpolarized 13C-Labeled Pyruvate Utilization as Methods for Detecting Tumor Response to Treatment

    Directory of Open Access Journals (Sweden)

    Timothy H. Witney

    2009-06-01

    Full Text Available Detection of early tumor responses to treatment can give an indication of clinical outcome. Positron emission tomography measurements of the uptake of the glucose analog, [18F] 2-fluoro-2-deoxy-d-glucose (FDG, have demonstrated their potential for detecting early treatment response in the clinic. We have shown recently that 13C magnetic resonance spectroscopy and spectroscopic imaging measurements of the uptake and conversion of hyperpolarized [1-13C]pyruvate into [1-13C]lactate can be used to detect treatment response in a murine lymphoma model. The present study compares these magnetic resonance measurements with changes in FDG uptake after chemotherapy. A decrease in FDG uptake was found to precede the decrease in flux of hyperpolarized 13C label between pyruvate and lactate, both in tumor cells in vitro and in tumors in vivo. However, the magnitude of the decrease in FDG uptake and the decrease in pyruvate to lactate flux was comparable at 24 hours after drug treatment. In cells, the decrease in FDG uptake was shown to correlate with changes in plasma membrane expression of the facilitative glucose transporters, whereas the decrease in pyruvate to lactate flux could be explained by an increase in poly(ADP-ribose polymerase activity and subsequent depletion of the NAD(H pool. These results show that measurement of flux between pyruvate and lactate may be an alternative to FDG-positron emission tomography for imaging tumor treatment response in the clinic.

  8. Quantitative measurement of exchange dynamics in proteins via {sup 13}C relaxation dispersion of {sup 13}CHD{sub 2}-labeled samples

    Energy Technology Data Exchange (ETDEWEB)

    Rennella, Enrico; Schuetz, Anne K.; Kay, Lewis E., E-mail: kay@pound.med.utoronto.ca [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)

    2016-06-15

    Methyl groups have emerged as powerful probes of protein dynamics with timescales from picoseconds to seconds. Typically, studies involving high molecular weight complexes exploit {sup 13}CH{sub 3}- or {sup 13}CHD{sub 2}-labeling in otherwise highly deuterated proteins. The {sup 13}CHD{sub 2} label offers the unique advantage of providing {sup 13}C, {sup 1}H and {sup 2}H spin probes, however a disadvantage has been the lack of an experiment to record {sup 13}C Carr–Purcell–Meiboom–Gill relaxation dispersion that monitors millisecond time-scale dynamics, implicated in a wide range of biological processes. Herein we develop an experiment that eliminates artifacts that would normally result from the scalar coupling between {sup 13}C and {sup 2}H spins that has limited applications in the past. The utility of the approach is established with a number of applications, including measurement of ms dynamics of a disease mutant of a 320 kDa p97 complex.

  9. Selective release of D and 13C from insoluble organic matter of the Murchison meteorite by impact shock

    Science.gov (United States)

    Mimura, Koichi; Okamoto, Michioki; Sugitani, Kenichiro; Hashimoto, Shigemasa

    2007-03-01

    We performed shock-recovery experiments on insoluble organic matter (IOM) purified from the Murchison meteorite, and determined the abundances and isotope ratios of hydrogen and carbon in the shocked IOM sample. We also performed shock experiments on type III kerogen and compared the results of these experiments with the experimental results regarding IOM.The shock selectively released D and 13C from the IOM, while it preferably released H and 12C from the kerogen. The release of these elements from IOM cannot be explained in terms of the isotope effect, whereas their release from kerogen can be explained by this effect. The selective release of heavier isotopes from IOM would be due to its structure, in which D and 13C-enriched parts are present as an inhomogeneity and are weakly attached to the main network. Shock gave rise to a high release of D even at a lower degree of dehydrogenation compared with the stepwise heating of IOM. This effective release of D is probably an inherent result of shock, in which a dynamic high-pressure and high-temperature condition prevails. Thus, shock would effectively control the hydrogen isotope behavior of extraterrestrial organic matter during the evolution of the solar nebula.

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

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2011-01-01

    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...

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Science.gov (United States)

    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

  13. Processing of 13C-labelled phytoplankton in a fine-grained sandy-shelf sediment (North Sea): relative importance of different macrofauna species

    DEFF Research Database (Denmark)

    Kamp, Anja; Witte, Ursula

    2005-01-01

    by additional laboratory experiments on the role of the dominant macrofauna organism, the bivalve Fabulina fabula (Bivalvia: Tellinidae), for particulate organic matter subduction to deeper sediment layers. The specific uptake of algal 13C by macrofauna organisms was visible after 12 h and constantly increased...... carbon processing. Predatory macrofauna organisms like Nephtys spp. (Polychaeta: Nephtyidae) also quickly became labelled. The rapid subduction of fresh organic matter by F. fabula down to ca. 4 to 7 cm sediment depth could be demonstrated, and it is suggested that entrainment by macrofauna in this fine...

  14. 13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans.

    Directory of Open Access Journals (Sweden)

    Blair C R Dancy

    Full Text Available 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.

  15. Spectral fitting for signal assignment and structural analysis of uniformly {sup 13}C-labeled solid proteins by simulated annealing based on chemical shifts and spin dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Matsuki, Yoh; Akutsu, Hideo; Fujiwara, Toshimichi [Osaka University, Institute for Protein Research (Japan)], E-mail: tfjwr@protein.osaka-u.ac.jp

    2007-08-15

    We describe an approach for the signal assignment and structural analysis with a suite of two-dimensional {sup 13}C-{sup 13}C magic-angle-spinning solid-state NMR spectra of uniformly {sup 13}C-labeled peptides and proteins. We directly fit the calculated spectra to experimental ones by simulated annealing in restrained molecular dynamics program CNS as a function of atomic coordinates. The spectra are calculated from the conformation dependent chemical shift obtained with SHIFTX and the cross-peak intensities computed for recoupled dipolar interactions. This method was applied to a membrane-bound 14-residue peptide, mastoparan-X. The obtained C', C{sup {alpha}} and C{sup {beta}} chemical shifts agreed with those reported previously at the precisions of 0.2, 0.7 and 0.4 ppm, respectively. This spectral fitting program also provides backbone dihedral angles with a precision of about 50 deg. from the spectra even with resonance overlaps. The restraints on the angles were improved by applying protein database program TALOS to the obtained chemical shifts. The peptide structure provided by these restraints was consistent with the reported structure at the backbone RMSD of about 1 A.

  16. 13C-labeled 18 : 2n-6 recovered in brush border membrane phospholipids short time after administration

    DEFF Research Database (Denmark)

    Vistisen, Bodil; Høy, Carl-Erik

    2004-01-01

    fatty acids in the two phospholipid pools. Minor effects on BBM-PC and BBM-PE fatty acid profiles (mole-%) were observed. The present study demonstrated for the first time incorporation of C-13-labeled 18:2n-6 into BBM-PC 2 hours and 6 hours after intragastric administration of L*L*L* or ML......*M. This emphasizes the influence of the dietary fatty acid on BBM fatty acid composition and the rapid incorporation of dietary long-chain fatty acids into intestinal enterocyte phospholipids. Medium-chain fatty acids in a single meal exert only a minor influence on the BBM phospholipid fatty acid profile....

  17. Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

    Science.gov (United States)

    Váradi, Csaba; Mittermayr, Stefan; Millán-Martín, Silvia; Bones, Jonathan

    2016-12-01

    Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13 C 6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12 C 6 'light' and 13 C 6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

  18. A study of lignin degradation in leaf and needle litter using 13C-labelled tetramethylammonium hydroxide (TMAH) thermochemolysis: comparison with CuO oxidation and van Soest methods.

    NARCIS (Netherlands)

    Klotzbücher, T.; Filley, T.R.; Kaiser, K.; Kalbitz, K.

    2011-01-01

    We studied the degradation of lignin in leaf and needle litter of ash, beech, maple, pine and spruce using 13C-labelled tetramethylammonium hydroxide (13C TMAH) thermochemolysis. Samples were allowed to decompose for 27 months in litter bags at a German spruce forest site, resulting in a range of

  19. Native and mass labeled [13C14]-decabromodiphenylethane (DBDPE). Characterization and use in determination of DBDPE in sewage sludge

    Energy Technology Data Exchange (ETDEWEB)

    McCrindle, R. [Guelph Univ., ON (Canada). Chemistry Dept.; Chittim, B.; Konstantinov, A.; McAlees, A.; Potter, D.; Tashiro, C.; Yeo, B. [Wellington Laboratories, Guelph, ON (Canada); Kolic, T.; MacPherson, K.; Reiner, E. [Ontario Ministry of the Environment, Toronto, ON (Canada)

    2004-09-15

    Decabromodiphenylethane (DBDPE) is a relatively new brominated flame retardant for use in polystyrene and polyolefin-based thermoplastic formulations. DBDPE was introduced in the early 90's, and is marketed as a non-diphenyl oxide based alternative to brominated diphenyl ethers (BDE)i. One study has shown that DBDPE produces no dioxins and only minor quantities of 2,3,7,8-TBDF under pyrolysis conditions. Although currently DBDPE is not as widely used as decabromodiphenyl ether (BDE-209) because of the higher cost, it is predicted that DBDPE will soon become one of the major flame retardants used by the thermoplastics industry. The presence of DBDPE in sediments, sewage sludge and air samples was reported during Dioxin 2003. The levels of DBDPE found in sewage sludge ranged from 33 to approximately 100 ng/g dry weight, and its ratio to BDE-209 levels in quantified samples was 0.01 in the sediment and 0.3 to 0.6 in the sewage sludge samples examined. The lack of a surrogate standard for DBDPE definitely poses difficulties in determining levels accurately. This study has two goals: the characterization of mass-labeled DBDPE and an assessment of its utility in determining DBDPE levels in environmental samples.

  20. Biogenic volatile organic compound and respiratory CO2 emissions after 13C-labeling: online tracing of C translocation dynamics in poplar plants.

    Science.gov (United States)

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

    2011-02-28

    Globally plants are the primary sink of atmospheric CO(2), 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. 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 (13)CO(2) to leaves or (13)C-glucose to shoots via xylem uptake. The translocation of (13)CO(2) from the source to other plant parts could be traced by (13)C-labeled isoprene and respiratory (13)CO(2) emission. In intact plants, assimilated (13)CO(2) 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). (13)C 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 CO(2), to a minor extent from xylem-transported sugars (7-11%) and from photosynthetic intermediates with slower turnover rates (8-11%). We quantified the plants' C loss as respiratory CO(2) and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux.

  1. Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: Application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins

    Energy Technology Data Exchange (ETDEWEB)

    Liu Aizhuo; Hu Weidong; Qamar, Seema; Majumdar, Ananya [Memorial Sloan-Kettering Cancer Center, Cellular Biochemistry and Biophysics Program (United States)

    2000-05-15

    In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in {sup 13}C/{sup 15}N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen-carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond {sup 3h}J{sub NC'} couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.

  2. Intraseasonal carbon sequestration and allocation in larch trees growing on permafrost in Siberia after 13C labeling (two seasons of 2013-2014 observation).

    Science.gov (United States)

    Masyagina, Oxana; Prokushkin, Anatoly; Kirdyanov, Alexander; Artyukhov, Aleksey; Udalova, Tatiana; Senchenkov, Sergey; Rublev, Aleksey

    2016-12-01

    This research is an attempt to study seasonal translocation patterns of photoassimilated carbon within trees of one of the high latitudes widespread deciduous conifer species Larix gmelinii (Rupr. Rupr). For this purpose, we applied whole-tree labeling by 13 CO 2 , which is a powerful and effective tool for tracing newly developed assimilates translocation to tissues and organs of a tree. Experimental plot has been established in a mature 105-year-old larch stand located within the continuous permafrost area near Tura settlement (Central Siberia, 64°17'13″N, 100°11'55″E, 148 m a.s.l.). Measurements of seasonal photosynthetic activity and foliage parameters (i.e., leaf length, area, biomass, etc.), and sampling were arranged from early growing season (June 8, 2013; May 14, 2014) until yellowing and senescence of needles (September 17, 2013; September 14, 2014). Labeling by 13 C of the tree branch (June 2013, for 3 branch replicates in 3 different trees) and the whole tree was conducted at early (June 2014), middle (July 2014), and late (August 2013) phase of growing season (for different trees in 3 replicates each time) by three pulses [(CO 2 )max = 3000-4000 ppmv, 13 CO 2 (30 % v/v)]. We found at least two different patterns of carbon translocation associated with larch CO 2 assimilation depending on needle phenology. In early period of growing season (June), 13 C appearing in newly developed needles is a result of remobilized storage material use for growth purposes. Then approximately at the end of June, growth processes is switching to storage processes lasting to the end of growing season.

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

    Science.gov (United States)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

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

  4. Qualitative metabolome analysis of human cerebrospinal fluid by 13C-/12C-isotope dansylation labeling combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

    Science.gov (United States)

    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 (13)C-dansyl and (12)C-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. © American Society for Mass Spectrometry, 2011

  5. XRD and solid state 13C-NMR evaluation of the crystallinity enhancement of 13C-labeled bacterial cellulose biosynthesized by Komagataeibacter xylinus under different stimuli: A comparative strategy of analyses.

    Science.gov (United States)

    Meza-Contreras, Juan C; Manriquez-Gonzalez, Ricardo; Gutiérrez-Ortega, José A; Gonzalez-Garcia, Yolanda

    2018-05-22

    The production and crystallinity of 13 C bacterial cellulose (BC) was examined in static culture of Komagataeibacter xylinus with different chemical and physical stimuli: the addition of NaCl or cloramphenicol as well as exposure to a magnetic field or to UV light. Crystalline BC biosynthesized under each stimulus was studied by XRD and solid state 13 C NMR analyses. All treatments produced BC with enhanced crystallinity over 90% (XRD) and 80% (NMR) compared to the control (83 and 76%, respectively) or to Avicel (77 and 62%, respectively). The XRD data indicated that the crystallite size was 80-85 Å. Furthermore, changes on the allomorphs (I α and I β ) ratio tendency of BC samples addressed to the stimuli were estimated using the C4 signal from 13 C NMR data. These results showed a decrease of the allomorph I α (3%) when BC was biosynthesized with UV light and chloramphenicol compared to control (58.79%). In contrast, the BC obtained with NaCl increased up to 60.31% of the I α allomorph ratio. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Direct monitoring by carbon-13 nuclear magnetic resonance spectroscopy of the metabolism and metabolic rate of 13C-labeled compounds in vivo.

    Science.gov (United States)

    Iida, K; Hidoh, O; Fukami, J; Kajiwara, M

    1991-01-01

    Carbon-13 nuclear magnetic resonance spectroscopy has been used to observe the transformations of [1-13C]-D-glucose to [1,1'-13C2]-D-trehalose, and [3-13C]-L-alanine to [2-13C]-L-glutamic acid in the living body of Gryllodes sigillatus. [3-13C]-D-Alanine was not metabolized. The metabolic rate of [1-13C]-D-glucose was found to be altered by prior injection of boric acid.

  7. Clonal variability for water use efficiency and carbon isotope discrimination ( 13C) in selected clones of a few Eucalyptus species

    CSIR Research Space (South Africa)

    Mohan Raju, B

    2011-11-01

    Full Text Available and develop high water use efficient clones to cultivate under water limited environments. The major objective was to assess the eucalyptus clones for variability in WUE and to determine the relationship between WUE and carbon isotope discrimination ( 13C...

  8. Investigation of the biosynthesis of acetyl-CoA and oxaloacetic acid from pyruvic acid and the quantitative evaluation of incorporated 13C-labeled l-alanine in Arthrobacter hyalinus

    International Nuclear Information System (INIS)

    Katsumi Iida

    2014-01-01

    Studies on the contribution to acetyl-CoA and oxaloacetic acid from the pyruvic acid transformation from l-alanine in Arthrobacter hyalinus were conducted by means of feeding experiments with l-[1- 13 C]alanine and l-[3- 13 C]alanine, followed by an analysis of the labeling patterns of coproporphyrinogen III using 13 C NMR spectroscopy. The results demonstrated that l-alanine was transformed via pyruvic acid to both acetyl-CoA and oxaloacetic acid. Additionally, the quantitative analysis indicated that pyruvic acid was transformed to acetyl-CoA and oxaloacetic acid in the ratio of 1:0.8. (author)

  9. NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23-230)

    International Nuclear Information System (INIS)

    Liu Aizhuo; Riek, Roland; Wider, Gerhard; Schroetter, Christine von; Zahn, Ralph; Wuethrich, Kurt

    2000-01-01

    A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly 15 N, 13 C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable 15 N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse 15 N relaxation rates of unfolded polypeptides in high resolution constant-time [ 1 H, 15 N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the 15 N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence

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

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  11. Incorporation of 13C labeled Pinus ponderosa needle and fine root litter into soil organic matter measured by Py-GC/MS-C-IRMS

    Science.gov (United States)

    Mambelli, S.; Gleixner, G.; Dawson, T. E.; Bird, J. A.; Torn, M. S.

    2006-12-01

    Developing effective strategies for enhancing C storage in soils requires understanding the influence of plant C quality. In turn, plant C quality impacts the decay continuum between plant residue and humified, stable SOM. This remains one of the least understood aspects of soil biogeochemistry. We investigated the initial phase of incorporation of 13C labeled Pinus ponderosa needle and fine root litter into SOM. The two litter types were placed in separate microcosms in the A horizon in a temperate conifer soil. Curie-point pyrolysis-gas chromatography coupled with on-line mass spectrometry and isotope ratio mass spectrometry (Py-GC/MS-C- IRMS) were used to determine the identity and the 13C enrichment of pyrolysis products (fragments of carbohydrates, lignin, proteins and lipids). We compared the two initial litter types, needles and fine roots, to samples of the bulk soil (A horizon, < 2mm) and soil humin fraction (from chemical solubility) obtained from each microcosm 1.5y after litter addition. Pyrolysis of plant material and SOM produced 56 suitable products for isotopic analysis; of them, 15 occurred in both the litter and bulk soil, 7 in both the litter and the humin fraction and 9 in both bulk soil and the humin fraction. The pyrolysis products found in common in the plant and soil were related either to polysaccharides or were non-specific and could have originated from various precursors. The data suggest that the majority of plant inputs, both from needles or fine roots, were degraded very rapidly. In the humin fraction, the most recalcitrant pool of C in soil, with a measured turnover time of 260y (this soil), only products from the fragmentation of polysaccharides and alkyl-benzene compounds were found. Comparisons of the enrichment normalized by input level suggest little difference between the incorporation of C from needles versus fine roots into SOM. The most enriched fragments in the humin fraction were products from polysaccharides degradation

  12. Structure of Selected Derivates of p-Hydroxy Cynamonic Acid According to 13C CP MAS NMR and DFT Calculation

    International Nuclear Information System (INIS)

    Pisklak, D.M.; Wawer, I.; Tkaczyk, M.

    2005-01-01

    Derivatives of p-hydroxy cynamonic acid are widely occurring in fruits, vegetables, tea and coffee. They exhibit strong antioxidant activity due to the presence of phenolic group. Epidemiological, biological and biochemical data support health beneficial role of this group of compounds and anticarcinogenic, antimutagenic and antiinflamatory effects have been reported. The most common caffeic acid contributes significantly to the total polyphenol intake and has been suggested to play a role in the apparent association between the regular consumption of polyphenol-rich food and beverages, and the prevetion of inflammatory and proliferative diseases. 13 C MAS NMR spectra were recorded on a BRUKER DSX 400 spectrometer at 400,13. Powder samples were spun in a 4 mm rotor at 10 kHz ( 13 C). Signals were assigned:- By comparison with solution spectra; - Using dipolar dephasing and variable contact time experiments; - Confirmed by DFT calculations of shielding constants. The differences in chemical shifts between solution and solid state spectra are due to the formation of intramolecular and intermolecular hydrogen bonds, including C-OH...OC within cyclic dimers. (author)

  13. Genetic algorithm as a variable selection procedure for the simulation of 13C nuclear magnetic resonance spectra of flavonoid derivatives using multiple linear regression.

    Science.gov (United States)

    Ghavami, Raoof; Najafi, Amir; Sajadi, Mohammad; Djannaty, Farhad

    2008-09-01

    In order to accurately simulate (13)C NMR spectra of hydroxy, polyhydroxy and methoxy substituted flavonoid a quantitative structure-property relationship (QSPR) model, relating atom-based calculated descriptors to (13)C NMR chemical shifts (ppm, TMS=0), is developed. A dataset consisting of 50 flavonoid derivatives was employed for the present analysis. A set of 417 topological, geometrical, and electronic descriptors representing various structural characteristics was calculated and separate multilinear QSPR models were developed between each carbon atom of flavonoid and the calculated descriptors. Genetic algorithm (GA) and multiple linear regression analysis (MLRA) were used to select the descriptors and to generate the correlation models. Analysis of the results revealed a correlation coefficient and root mean square error (RMSE) of 0.994 and 2.53ppm, respectively, for the prediction set.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wiedemann, Christoph; Goradia, Nishit; Häfner, Sabine [Leibniz Institute for Age Research, Fritz Lipmann Institute, Research Group Biomolecular NMR Spectroscopy (Germany); Herbst, Christian [Ubon Ratchathani University, Department of Physics, Faculty of Science (Thailand); Görlach, Matthias; Ohlenschläger, Oliver; Ramachandran, Ramadurai, E-mail: raman@fli-leibniz.de [Leibniz Institute for Age Research, Fritz Lipmann Institute, Research Group Biomolecular NMR Spectroscopy (Germany)

    2015-10-15

    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 ({sup 13}C, {sup 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 {sup 1}J{sub CαN} and {sup 2}J{sub CαN} couplings to transfer the {sup 15}N{sub x} magnetisation from amino acid residue ‘i’ to adjacent residues via the application of a band-selective {sup 15}N–{sup 13}C{sup 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.

  15. Synthesis of 24-methyl sterols sterospecifically labelled with 2H in the isopropyl methyl groups. 13C NMR spectral assignment of C-26 and C-27 resonances

    International Nuclear Information System (INIS)

    Colombo, D.; Ronchetti, F.; Toma, L.

    1990-01-01

    Through analysis of the 13 C NMR spectra of (25S)-[27- 2 H]campesterol (1) and (25R)-[26- 2 H]dihydrobrassicasterol (2), the C-26 and C-27 resonances have been unambiguously assigned; the biosynthetic applications are discussed. (author)

  16. Synthesis of specifically 15N- and 13C-labeled antitumor (2-Haloethyl)nitrosoureas. The study of their conformations in solution by nitrogen-15 and carbon-13 nuclear magnetic resonance and evidence for stereoelectronic control in their aqueous decomposition

    International Nuclear Information System (INIS)

    Lown, J.W.; Chauhan, S.M.S.

    1981-01-01

    The synthesis of certain specifically 15 N, 13 C, and 2 H isotope labeled 1-(2-chloroethyl)-3-alkyl-1-nitrosoureas (CENUs) is described. Spectroscopic examination of CENUs and their isotope-labeled counterparts by 1 H, 15 N, and 13 C NMR and infrared spectra indicates that they adopt preferred conformations in nonpolar aprotic solvents in which the NO group is aligned toward the 2-chloroethyl group. The result is in accord with the conformation of MeCCNU in the crystalline state derived from X-ray diffraction. The chemical shifts and coupling constants in the CENUs change with both solvent polarity and basicity. In aqueous phosphate buffer there is evidence for the formation of a tetrahedral intermediate, the conformation of which alters according to the reaction conditions and ultimately controls the formation of the aqueous decomposition products of CENUs. This is revealed most clearly by 13 C NMR of carbonyl- 13 C- and nitroso- 15 N-labeled BCNU and CCNU where two distinct 15 N-coupled 13 C doublets with different chemical shifts are observed. The rate of conformational change is comparable with the rate of decomposition of CENUs (via the second conformer) and may therefore represent the critical initial step of the latter process in vivo. The intermediacy of the postulated tetrahedral intermediates for CENUs is supported by observed 18 O exchange into the carbonyl group in 18 O-enriched water. Consideration of the conformations of the intermediates and of the alignment of the heteroatom lone pairs provides a satisfactory interpretation of the reactions of CENUs in aqueous solution as well as their pH dependence in terms of strict steroelectronic control and accounts for the formation of the observed products

  17. 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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

  18. Synthesis of [21-13C]-cholesterol

    International Nuclear Information System (INIS)

    Caballero, G.M.; Gros, E.G.

    1994-01-01

    The synthesis of [21- 13 C]-cholesterol from 3β-O-(t-butyldimethylsilyl)-17β-cyano-androst-5-ene is described. Labelled carbon-atom was introduced by Grignard reaction of nitrile derivative with [ 13 C]-methylmagnesium iodide. Location of label was confirmed by 13 C-NMR spectroscopy. (author)

  19. Selection of drought tolerant and high water use efficient rice cultivars through /sup 13/C isotope discrimination technique

    International Nuclear Information System (INIS)

    Akhtar, J.; Sabir, S.A.; Ashraf, M.Y.; Monneveux, P.; Serraj, R.

    2010-01-01

    Carbon isotope discrimination ('A') has been suggested as an indirect tool for selecting plants having higher water use efficiency (WUE) and yield potential. Enhancing WUE is an important breeding objective as water scarcity is increasing with every passing day. This study was undertaken to assess the genotypic variation and relationship between leaf, straw, grain 'A', grain yield and WUE in eight aromatic rice cultivars grown in lysimeters under three water regimes, in absence of drainage and runoff. Highly significant positive correlations were found between aboveground biomass and WUEB, and grain yield and WUEG, due to the low variation in water consumed by different cultivars. Leaf, straw and grain A showed a consistent variation across treatments and cultivars. Under water stress conditions, both leaf and straw 'A' were positively correlated to grain yield and WUEG. In all the water treatments, WUEG was positively correlated to harvest index and negatively to plant height. All the mutants from Basmati 385 had significantly higher 'A' values as compared to the mutants from Basmati 370. It was concluded that the new cultivar, Basmati 385, represents a better genetic source for 'A' improvement than the old cultivar, Basmati 370. (author)

  20. Synthesis and applications of 13C glycerol

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  1. Stereoselective synthesis of L-[4-13C]carnitine

    International Nuclear Information System (INIS)

    Unkefer, C.J.; Ehler, D.S.

    1991-01-01

    The stereoselective synthesis of L-[4- 13 C]carnitine was achieved in 5 steps. The label was introduced from K 13 CN into an easily separated diastereomeric pair of 3-deoxy-D-[1- 13 C]aldohexoses. Reductive amination of the labeled aldohexose yielded the corresponding D-1-(dimethylamino)[1- 13 C]alditol which was oxidized in two steps and alkylated with iodomethane to yield L-[4- 13 C]carnitine. The stereochemical integrity at C-2 of the 3-deoxy-D-[1- 13 C]glucose precursor was maintained throughout the synthesis of L-[4- 13 C]carnitine. (author)

  2. Synthesis and applications of 13C glycerol

    International Nuclear Information System (INIS)

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

    1994-01-01

    The authors are currently developing new synthetic routes to the various isotopomers of glycerol. Labeled glycerol is useful for 13 C enrichment of biomolecules. However, traditional methods of glycerol synthesis are not easily adapted for isotopic enrichment or have poor overall yields (12-15%). In addition, the use of glycerol for enrichment can be prohibitively expensive and its availability depends on the level of demand. The authors have developed a short de novo synthesis of [U- 13 C]glycerol from carbon dioxide (∼53% overall yield for four steps) and are currently examining the feasibility of synthesizing site-specific 13 C labeled glycerol and dihydroxyacetone (DHA) from methanol and carbon dioxide. The authors have examined the enzymatic conversion of [U- 13 C]glycerol to glyceraldehyde-3-monophosphate or dihydroxyacetone monophosphate (DHAP) with yields ranging from 25-50% (as determined by NMR spectroscopy). The authors are also pursuing the chemical conversion of 13 C labeled DHA to DHAP and the results are presented. Labeled DHAP is a possible enzymatic precursor for both labeled 2-deoxyribose and 2-deoxyribonucleic acids

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

    Science.gov (United States)

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

    2016-03-25

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Takeuchi, Koh; Gal, Maayan [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States); Takahashi, Hideo; Shimada, Ichio [National Institute of Advanced Industrial Science and Technology, Biomedicinal Information Research Center (Japan); Wagner, Gerhard, E-mail: gerhard_wagner@hms.harvard.edu [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States)

    2011-01-15

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

  5. Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection

    DEFF Research Database (Denmark)

    Weininger, Ulrich; Brath, Ulrika; Modig, Kristofer

    2014-01-01

    Protein dynamics on the microsecond-millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications...... on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the backbone amides or side-chain methyl groups, while experiments targeting other sites have been used more sparingly. Aromatic side chains are useful probes of protein dynamics, because...... they are over-represented in protein binding interfaces, have important catalytic roles in enzymes, and form a sizable part of the protein interior. Here we present an off-resonance R 1ρ experiment for measuring microsecond to millisecond conformational exchange of aromatic side chains in selectively (13)C...

  6. Accurate Determination of Leucine and Valine Side-chain Conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] Isotope Labeling, NOE Pattern Recognition, and Methine Cγ-Hγ/Cβ-Hβ Residual Dipolar Couplings

    International Nuclear Information System (INIS)

    Tang, Chun; Iwahara, Junji; Clore, G. Marius

    2005-01-01

    An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a 15 N/ 13 C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)-H N (i) and Hγ(i)-H N (i+1) NOEs in a 3D 15 N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine 13 C- 1 H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA Chitobiose , a 34 kDa homotrimeric phosphotransferase protein

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

    International Nuclear Information System (INIS)

    Pahl-Wostl, C.; Seelig, J.

    1986-01-01

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

  8. Flux response of glycolysis and storage metabolism during rapid feast/famine conditions in Penicillium chrysogenum using dynamic (13)C labeling.

    Science.gov (United States)

    de Jonge, Lodewijk; Buijs, Nicolaas A A; Heijnen, Joseph J; van Gulik, Walter M; Abate, Alessandro; Wahl, S Aljoscha

    2014-03-01

    The scale-up of fermentation processes frequently leads to a reduced productivity compared to small-scale screening experiments. Large-scale mixing limitations that lead to gradients in substrate and oxygen availability could influence the microorganism performance. Here, the impact of substrate gradients on a penicillin G producing Penicillium chrysogenum cultivation was analyzed using an intermittent glucose feeding regime. The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 μM down to 6.5 μM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100-fold changes. The intracellular flux changes were estimated on the basis of dynamic (13) C mass isotopomer measurements during three cycles of feast and famine using a novel hybrid modeling approach. The flux estimations indicated a high turnover of internal and external storage metabolites in P. chrysogenum under feast/famine conditions. The synthesis and degradation of storage requires cellular energy (ATP and UTP) in competition with other cellular functions including product formation. Especially, 38% of the incoming glucose was recycled once in storage metabolism. This result indicated that storage turnover is increased under dynamic cultivation conditions and contributes to the observed decrease in productivity compared to reference steady-state conditions. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Efficient Multi-Label Feature Selection Using Entropy-Based Label Selection

    Directory of Open Access Journals (Sweden)

    Jaesung Lee

    2016-11-01

    Full Text Available Multi-label feature selection is designed to select a subset of features according to their importance to multiple labels. This task can be achieved by ranking the dependencies of features and selecting the features with the highest rankings. In a multi-label feature selection problem, the algorithm may be faced with a dataset containing a large number of labels. Because the computational cost of multi-label feature selection increases according to the number of labels, the algorithm may suffer from a degradation in performance when processing very large datasets. In this study, we propose an efficient multi-label feature selection method based on an information-theoretic label selection strategy. By identifying a subset of labels that significantly influence the importance of features, the proposed method efficiently outputs a feature subset. Experimental results demonstrate that the proposed method can identify a feature subset much faster than conventional multi-label feature selection methods for large multi-label datasets.

  10. The use of {sup 13}C labelling of bacterial lipids in the characterisation of ambient methane-oxidising bacteria in soils

    Energy Technology Data Exchange (ETDEWEB)

    Crossman, Z.M.; Evershed, R.P. [Bristol Univ., Organic Geochemistry Unit, Biogeochemistry Research Centre, Bristol (United Kingdom); Ineson, P. [York Univ., Dept. of Biology, York (United Kingdom)

    2005-05-15

    The occurrence of methane-oxidising bacteria in soils has received increasing attention because of their role as a sink for atmospheric methane. However, such bacteria are not amenable to modern culturing techniques and hence the widespread interest in the development of methods of cultivation-independent analysis. In the following investigation, a combination of stable isotope labelling with phospholipid fatty acid (PLFA) and bacteriohopanoid analysis was employed in an effort to characterise this functional group of bacteria. Results suggest a novel population of methane-oxidising bacteria related to type II culturable methanotrophs, in particular, the Methylocapsa and Methylocella genera of bacteria. (Author)

  11. Changes in carbon uptake and allocation patterns in Quercus robur seedlings in response to elevated CO2 and water stress: an evaluation with 13C labelling

    International Nuclear Information System (INIS)

    Vivin, P.; Guehl, J.M.

    1997-01-01

    A semi-closed (CO2)-C-13 labelling system (1.5% C-13) was used to assess both carbon uptake and allocation within pedunculate oak seedlings (Quercus robur L) grown under ambient (350 vpm) and elevated (700 vpm) atmospheric CO2 concentration ([CO2]) and in either well-watered or droughted conditions. Pulse-chase C-13 labelling data highlighted the direct positive effect of elevated CO2 on photosynthetic carbon acquisition. Consequently, in well-watered conditions, CO2-enriched plants produced 1.52 times more biomass (dry mass at harvest) and 1.33 times more dry root matter (coarse plus fine roots) over the 22-week growing period than plants grown under ambient [CO2]. The root/shoot biomass ratio was decreased both by drought and [CO2], despite lower N concentrations in CO2-enriched plants. However, both long-term and short-term C allocation to fine roots were not altered by CO2, and relative specific allocation (RSA), a parameter expressing sink strength, was hip her in all plant organs under 700 vpm compared to 350 vpm. Results showed that C availability for growth and metabolic processes was greater in fine roots of oaks grown under an elevated CO2 atmosphere irrespective of soil water availability [fr

  12. Optimized slice-selective 1H NMR experiments combined with highly accurate quantitative 13C NMR using an internal reference method

    Science.gov (United States)

    Jézéquel, Tangi; Silvestre, Virginie; Dinis, Katy; Giraudeau, Patrick; Akoka, Serge

    2018-04-01

    Isotope ratio monitoring by 13C NMR spectrometry (irm-13C NMR) provides the complete 13C intramolecular position-specific composition at natural abundance. It represents a powerful tool to track the (bio)chemical pathway which has led to the synthesis of targeted molecules, since it allows Position-specific Isotope Analysis (PSIA). Due to the very small composition range (which represents the range of variation of the isotopic composition of a given nuclei) of 13C natural abundance values (50‰), irm-13C NMR requires a 1‰ accuracy and thus highly quantitative analysis by 13C NMR. Until now, the conventional strategy to determine the position-specific abundance xi relies on the combination of irm-MS (isotopic ratio monitoring Mass Spectrometry) and 13C quantitative NMR. However this approach presents a serious drawback since it relies on two different techniques and requires to measure separately the signal of all the carbons of the analyzed compound, which is not always possible. To circumvent this constraint, we recently proposed a new methodology to perform 13C isotopic analysis using an internal reference method and relying on NMR only. The method combines a highly quantitative 1H NMR pulse sequence (named DWET) with a 13C isotopic NMR measurement. However, the recently published DWET sequence is unsuited for samples with short T1, which forms a serious limitation for irm-13C NMR experiments where a relaxing agent is added. In this context, we suggest two variants of the DWET called Multi-WET and Profiled-WET, developed and optimized to reach the same accuracy of 1‰ with a better immunity towards T1 variations. Their performance is evaluated on the determination of the 13C isotopic profile of vanillin. Both pulse sequences show a 1‰ accuracy with an increased robustness to pulse miscalibrations compared to the initial DWET method. This constitutes a major advance in the context of irm-13C NMR since it is now possible to perform isotopic analysis with high

  13. Biosynthetic preparation of L-[13C]- and [15N]glutamate by Brevibacterium flavum

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  14. Preparation of 15N-13C-fulminic acid

    International Nuclear Information System (INIS)

    Wilmes, R.; Winnewisser, M.

    1993-01-01

    The precursor for the title compound was prepared in a three-step synthesis. The 13 C-label was incorporated in the first step employing 2- 13 C-ethyl acetate and the 15 N-label in the last step, using 15 N-sodium nitrite. Upon pyrolysis the precursor forms three fragments, one of them being the title compound. (Author)

  15. Synthesis and characterization of "1"3C_3-tristearin

    International Nuclear Information System (INIS)

    Wu Hangyu; Lin Lin; Li Lei; Chen Dazhou

    2011-01-01

    A highly efficient synthesis of "1"3C_3 labeled triglycerides of stearic acids from "1"3C_3-glycerol and stearic acids, by immobilized lipase-catalyzed in solvent-free medium was described. The structure of the product were characterized by fourier transform infrared spectrum (FT-IR), nuclear magnetic resonance (NMR), mass spectra (MS). The results showed that triglycerides of stearic acids contained three "1"3C atoms. The isotope abundance of "1"3C_3-tristearin was more than 99% and the yield was 80% of "1"3C_3-tristearin through calculation. Chemical purity (> 98%) was obtained by differential scanning calorimetry (DSC). (authors)

  16. 13C NMR for the assessment of human brain glucose metabolism in vivo

    International Nuclear Information System (INIS)

    Beckman, N.; Seelig, J.; Turkalj, I.; Keller, U.

    1991-01-01

    Proton-decoupled 13 C NMR spectra of the human head were obtained during hyperglycemic glucose clamping using intravenous infusions of [1- 13 C]glucose in normal volunteers. In addition to 13 C signals of mobile lipids, a variety of new metabolite resonances could be resolved for the first time in the human brain. At an enrichment level of 20% [1- 13 C]glucose, the signals of α- and β-glucose at 92.7 and 96.6 ppm, respectively, could be detected in the human brain after only an infusion period of 15 minutes. The spatial localization of the different regions of interest was confirmed by 13 C NMR spectroscopic imaging with a time resolution of 9 minutes. Increasing the enrichment level to 99% [1- 13 C]glucose not only improved the time resolution but allowed the detection of metabolic breakdown products of [1- 13 C]glucose. The time course of 13 C label incorporation into the C 2 , C 3 , and C 4 resonances of glutamate/glutamine and into lactate could be recorded in the human brain. These results suggest the possibility of obtaining time-resolved, spatially selective, and chemically specific information on the human body

  17. Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

    Energy Technology Data Exchange (ETDEWEB)

    Eddy, Matthew T. [Massachusetts Institute of Technology, Department of Chemistry (United States); Belenky, Marina [Brandeis University, Department of Chemistry (United States); Sivertsen, Astrid C. [Massachusetts Institute of Technology, Francis Bitter Magnet Laboratory (United States); Griffin, Robert G. [Massachusetts Institute of Technology, Department of Chemistry (United States); Herzfeld, Judith, E-mail: herzfeld@brandeis.edu [Brandeis University, Department of Chemistry (United States)

    2013-10-15

    The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new 'redox' approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-{sup 13}C] acetate does not label {alpha} carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-{sup 13}C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra.

  18. The synthesis of 5'-[14C1] and 3a, 4-[13C2] labelled panadiplon (U-78875; 3-(5'-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)-imidazo-[1,5a]-quinoxalin-4(5H)-one)

    International Nuclear Information System (INIS)

    Ackland, M.J.; Howard, M.R.; Dring, L.G.

    1993-01-01

    5'-[ 14 C 1 ]Panadiplon was prepared in 3 steps starting from [ 14 C 1 ]cyclopropane carboxylic acid and 3-(5'-cyano-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)-imidazo-[1,5a] -quinoxalin-4(5H)-one. 3a, 4-[ 13 C 2 ]Panadiplon was prepared in two steps from 13 C 2 -oxalic acid and N-1-(1-methylethyl)-o-phenylenediamine. The position of labelling was confirmed by the appearance of two coupled resonances (J C-C =80.59 Hz) at 121.95 and 154.39 ppm in the assigned 13 C-NMR spectrum. (Author)

  19. Triple resonance experiments for the simultaneous correlation of H6/H5 and exchangeable protons of pyrimidine nucleotides in 13C,15N-labeled RNA applicable to larger RNA molecules

    International Nuclear Information System (INIS)

    Woehnert, Jens; Goerlach, Matthias; Schwalbe, Harald

    2003-01-01

    Triple-resonance two-dimensional H6/H5(C4N)H and C6/C5(C4N)H experiments are described that provide through-bond H6/H5 or C6/C5 to imino/amino correlations in pyrimidine bases in 13 C, 15 N-labeled RNA. The experiments simultaneously transfer H6/H5 magnetization by an INEPT step to the C6/C5 nuclei and by homonuclear CC- and heteronuclear CN-TOCSY steps via the intervening C4 nucleus to the N3/N4 nuclei and then by a reverse INEPT step to the imino/amino hydrogens. The sensitivity of these experiments is high as demonstrated using a 30-nucleotide pyrimidine rich RNA at a concentration of 0.9 mM at temperatures of 10 deg. C and 25 deg. C. This indicates the general applicability of the experiments and the possibility to obtain correlations for imino resonances in non-canonical regions of the target RNA

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

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W. [Abbott Laboratories, Abbott Park, IL (United States)

    1994-12-01

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

  1. Non-stationary (13)C-metabolic flux ratio analysis.

    Science.gov (United States)

    Hörl, Manuel; Schnidder, Julian; Sauer, Uwe; Zamboni, Nicola

    2013-12-01

    (13)C-metabolic flux analysis ((13)C-MFA) has become a key method for metabolic engineering and systems biology. In the most common methodology, fluxes are calculated by global isotopomer balancing and iterative fitting to stationary (13)C-labeling data. This approach requires a closed carbon balance, long-lasting metabolic steady state, and the detection of (13)C-patterns in a large number of metabolites. These restrictions mostly reduced the application of (13)C-MFA to the central carbon metabolism of well-studied model organisms grown in minimal media with a single carbon source. Here we introduce non-stationary (13)C-metabolic flux ratio analysis as a novel method for (13)C-MFA to allow estimating local, relative fluxes from ultra-short (13)C-labeling experiments and without the need for global isotopomer balancing. The approach relies on the acquisition of non-stationary (13)C-labeling data exclusively for metabolites in the proximity of a node of converging fluxes and a local parameter estimation with a system of ordinary differential equations. We developed a generalized workflow that takes into account reaction types and the availability of mass spectrometric data on molecular ions or fragments for data processing, modeling, parameter and error estimation. We demonstrated the approach by analyzing three key nodes of converging fluxes in central metabolism of Bacillus subtilis. We obtained flux estimates that are in agreement with published results obtained from steady state experiments, but reduced the duration of the necessary (13)C-labeling experiment to less than a minute. These results show that our strategy enables to formally estimate relative pathway fluxes on extremely short time scale, neglecting cellular carbon balancing. Hence this approach paves the road to targeted (13)C-MFA in dynamic systems with multiple carbon sources and towards rich media. © 2013 Wiley Periodicals, Inc.

  2. Synthesis of edatrexate (2-13C-glutamate)

    International Nuclear Information System (INIS)

    DeGraw, J.I.; Colwell, W.T.; Jue, Thomas

    1997-01-01

    The experimental antitumor drug Edatrexate, labeled with 99% 13 C at the 2-position of the glutamate acid group was required for 13 C-magnetic resonance spectroscopy studies in biological media. Coupling of 2,4-diamino-4-deoxy-10-ethyl-10-deazapteroic acid with diethyl L-2- 13 C-glutamate as promoted by BOP reagent afforded Edatrexate (2- 13 C-glu) diethyl ester in 60% yield following purification by column chromatography. Saponification by aqueous NaOH in 2-methoxyethanol gave the target molecule in 44% yield or 26% overall. (author)

  3. Synthesis of [5,6-13C2, 1-14C]olivetolic acid, methyl [1'-13C]olivetolate and [5,6-13C2, 1-14C]cannabigerolic acid

    International Nuclear Information System (INIS)

    Porwoll, J.P.; Leete, E.

    1985-01-01

    Potential advanced intermediates in the biosynthesis of delta 9 -tetrahydrocannabinol, the major psychoactive principle of marijuana, have been synthesized labeled with two contiguous 13 C atoms and 14 C. Methyl [5,6- 13 C 2 , 1- 14 C]olivetolate was prepared from lithium [ 13 C 2 ]acetylide and dimethyl [2- 14 C]malonate. Reaction with geranyl bromide afforded methyl [5,6- 13 C 2 , 1- 14 C]cannabigerolate, and hydrolysis of these methyl esters with lithium propyl mercaptide yielded the corresponding labeled acids. The 13 C- 13 C couplings observable in the 13 C NMR spectra of these 13 C-enriched compounds and their synthetic precursors are recorded. Methyl [1'- 14 C]olivetolate was prepared from 13 CO 2 to confirm assignments of the 13 C chemical shifts in the pentyl side chain of these compounds. (author)

  4. A double-tracer technique to characterize absorption, distribution, metabolism and excretion (ADME) of [14C]-basimglurant and absolute bioavailability after oral administration and concomitant intravenous microdose administration of [13C6]-labeled basimglurant in humans.

    Science.gov (United States)

    Guerini, Elena; Schadt, Simone; Greig, Gerard; Haas, Ruth; Husser, Christophe; Zell, Manfred; Funk, Christoph; Hartung, Thomas; Gloge, Andreas; Mallalieu, Navita L

    2017-02-01

    1. The emerging technique of employing intravenous microdose administration of an isotope tracer concomitantly with an [ 14 C]-labeled oral dose was used to characterize the disposition and absolute bioavailability of a novel metabotropic glutamate 5 (mGlu5) receptor antagonist under clinical development for major depressive disorder (MDD). 2. Six healthy volunteers received a single 1 mg [ 12 C/ 14 C]-basimglurant (2.22 MBq) oral dose and a concomitant i.v. tracer dose of 100 μg of [ 13 C 6 ]-basimglurant. Concentrations of [ 12 C]-basimglurant and the stable isotope [ 13 C 6 ]-basimglurant were determined in plasma by a specific LC/MS-MS method. Total [ 14 C] radioactivity was determined in whole blood, plasma, urine and feces by liquid scintillation counting. Metabolic profiling was conducted in plasma, urine, blood cell pellet and feces samples. 3. The mean absolute bioavailability after oral administration (F) of basimglurant was ∼67% (range 45.7-77.7%). The major route of [ 14 C]-radioactivity excretion, primarily in form of metabolites, was in urine (mean recovery 73.4%), with the remainder excreted in feces (mean recovery 26.5%). The median t max for [ 12 C]-basimglurant after the oral administration was 0.71 h (range 0.58-1.00) and the mean terminal half-life was 77.2 ± 38.5 h. Terminal half-life for the [ 14 C]-basimglurant was 178 h indicating presence of metabolites with a longer terminal half-life. Five metabolites were identified with M1-Glucuronide as major and the others in trace amounts. There was minimal binding of drug to RBCs. IV pharmacokinetics was characterized with a mean ± SD CL of 11.8 ± 7.4 mL/h and a Vss of 677 ± 229 L. 4. The double-tracer technique used in this study allowed to simultaneously characterize the absolute bioavailability and disposition characteristics of the new oral molecular entity in a single study.

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

    Science.gov (United States)

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

    2014-01-01

    The growing demand for (13) C-metabolic flux analysis ((13) C-MFA) in the field of metabolic engineering and systems biology is driving the need to rationalize expensive and time-consuming (13) C-labeling experiments. Experimental design is a key step in improving both the number of fluxes that can be calculated from a set of isotopic data and the precision of flux values. We present IsoDesign, a software that enables these parameters to be maximized by optimizing the isotopic composition of the label input. It can be applied to (13) C-MFA investigations using a broad panel of analytical tools (MS, MS/MS, (1) H NMR, (13) C NMR, etc.) individually or in combination. It includes a visualization module to intuitively select the optimal label input depending on the biological question to be addressed. Applications of IsoDesign are described, with an example of the entire (13) C-MFA workflow from the experimental design to the flux map including important practical considerations. IsoDesign makes the experimental design of (13) C-MFA experiments more accessible to a wider biological community. IsoDesign is distributed under an open source license at http://metasys.insa-toulouse.fr/software/isodes/ © 2013 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    Xu, Su; Shen, Jun

    2006-10-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Stocking, E.; Khalsa, O.; Martinez, R.A.; Silks, L.A. III [Los Alamos National Laboratory, NM (United States)

    1994-12-01

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

  8. Generation and relaxation of high rank coherences in AX3 systems in a selectively methionine labelled SH2 domain

    International Nuclear Information System (INIS)

    Kloiber, Karin; Fischer, Michael; Ledolter, Karin; Nagl, Michael; Schmid, Walther; Konrat, Robert

    2007-01-01

    The usefulness of selective isotope labelling patterns is demonstrated using the C-terminal SH2 domain of PLC-γ1 selectively 13 C labelled at methionine methyl groups. We demonstrate the generation and relaxation of coherences that are second rank in protons and first rank in carbons that derive from quadrupolar order in protons. The decay rates of second rank double quantum proton coherences are measured. These terms exhibit fewer channels for cross-correlated relaxation compared to single quantum coherences. Our results indicate the potential application of the measurement of high order proton coherences to the analysis of dynamics in methyl-bearing side chains

  9. Insight into the labeling mechanism of acceleration selective arterial spin labeling

    DEFF Research Database (Denmark)

    Schmid, Sophie; Petersen, Esben T; Van Osch, Matthias J P

    2017-01-01

    OBJECTIVES: Acceleration selective arterial spin labeling (AccASL) is a spatially non-selective labeling technique, used in traditional ASL methods, which labels spins based on their flow acceleration rather than spatial localization. The exact origin of the AccASL signal within the vasculature......-ASL, combined AccASL and VS-ASL signal, and signal from one module with crushing from the other. RESULTS: The label created with AccASL has an overlap of approximately 50% in the vascular region with VS-ASL, but also originates from smaller vessels closer to the capillaries. CONCLUSION: AccASL is able to label...

  10. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

    International Nuclear Information System (INIS)

    Miyanoiri, Yohei; Ishida, Yojiro; Takeda, Mitsuhiro; Terauchi, Tsutomu; Inouye, Masayori; Kainosho, Masatsune

    2016-01-01

    We recently developed a practical protocol for preparing proteins bearing stereo-selectively 13 C-methyl labeled leucines and valines, instead of the commonly used 13 C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

  11. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

    Energy Technology Data Exchange (ETDEWEB)

    Miyanoiri, Yohei [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ishida, Yojiro [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Terauchi, Tsutomu [Tokyo Metropolitan University, Graduate School of Science and Engineering (Japan); Inouye, Masayori [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2016-06-15

    We recently developed a practical protocol for preparing proteins bearing stereo-selectively {sup 13}C-methyl labeled leucines and valines, instead of the commonly used {sup 13}C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

  12. Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for 13C-MFA.

    Science.gov (United States)

    Kappelmann, Jannick; Klein, Bianca; Geilenkirchen, Petra; Noack, Stephan

    2017-03-01

    In recent years the benefit of measuring positionally resolved 13 C-labeling enrichment from tandem mass spectrometry (MS/MS) collisional fragments for improved precision of 13 C-Metabolic Flux Analysis ( 13 C-MFA) has become evident. However, the usage of positional labeling information for 13 C-MFA faces two challenges: (1) The mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) The positional identity of carbon atoms of product ions needs to be known. The present contribution addresses the latter challenge by deducing the maximal positional labeling information contained in LC-ESI-MS/MS spectra of product anions of central metabolism as well as product cations of amino acids. For this purpose, we draw on accurate mass spectrometry, selectively labeled standards, and published fragmentation pathways to structurally annotate all dominant mass peaks of a large collection of metabolites, some of which with a complete fragmentation pathway. Compiling all available information, we arrive at the most detailed map of carbon atom fate of LC-ESI-MS/MS collisional fragments yet, comprising 170 intense and structurally annotated product ions with unique carbon origin from 76 precursor ions of 72 metabolites. Our 13 C-data proof that heuristic fragmentation rules often fail to yield correct fragment structures and we expose common pitfalls in the structural annotation of product ions. We show that the positionally resolved 13 C-label information contained in the product ions that we structurally annotated allows to infer the entire isotopomer distribution of several central metabolism intermediates, which is experimentally demonstrated for malate using quadrupole-time-of-flight MS technology. Finally, the inclusion of the label information from a subset of these fragments improves flux precision in a Corynebacterium glutamicum model of the central carbon metabolism.

  13. Inter-laboratory comparison of elemental analysis and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Part I: delta13C measurements of selected compounds for the development of an isotopic Grob-test.

    Science.gov (United States)

    Serra, F; Janeiro, A; Calderone, G; Rojas, J M Moreno; Rhodes, C; Gonthier, L A; Martin, F; Lees, M; Mosandl, A; Sewenig, S; Hener, U; Henriques, B; Ramalho, L; Reniero, F; Teixeira, A J; Guillou, C

    2007-03-01

    This study was directed towards investigating suitable compounds to be used as stable isotope reference materials for gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) calibration. Several compounds were selected from those used in the 'Grob-test' mixture. Oxygen- and nitrogen-containing substances were added to these compounds to allow the mixture to be used as a possible multi-isotopic calibration tool for 2H/1H, 13C/12C, 15N/14N and 18O/16O ratio determinations. In this paper we present the results of delta13C measurements performed by the consortium of the five laboratories taking part in this inter-calibration exercise. All the compounds were individually assessed for homogeneity, short-term stability and long-term stability by means of EA-IRMS, as required by the bureau communitaire de reference (BCR) Guide for Production of Certified Reference Materials. The results were compared then with the GC-C-IRMS measurements using both polar and non-polar columns, and the final mixture of selected compounds underwent a further certification exercise assessing limits of accuracy and reproducibility under specified GC-C-IRMS conditions. Copyright 2007 John Wiley & Sons, Ltd.

  14. Iminium ion chemistry of mitosene DNA alkylating agents. Enriched 13C NMR and isolation studies.

    Science.gov (United States)

    Ouyang, A; Skibo, E B

    2000-05-16

    Described herein is a study of the reductive alkylation chemistry of mitosene antitumor agents. We employed a 13C-enriched electrophilic center to probe the fate of the iminium ion resulting from reductive activation. The 13C-labeled center permitted the identification of complex products resulting from alkylation reactions. In the case of DNA reductive alkylation, the type and number of alkylation sites were readily assessed by 13C NMR. Although there has been much excellent work done in the area of mitosene chemistry and biochemistry, the present study provides a number of new findings: (1) The major fate of the iminium ion is head-to-tail polymerization, even in dilute solutions. (2) Dithionite reductive activation results in the formation of mitosene sulfite esters as well as the previously observed sulfonate adducts. (3) The mitosene iminium ion alkylates the adenosine 6-amino group as well as the guanosine 2-amino group. The identification of the latter adduct was greatly facilitated by the 13C-label at the electrophilic center. (4) The mitosene iminium ion alkylates DNA at both nitrogen and oxygen centers without any apparent base selectivity. The complexity of mitosene reductive alkylation of DNA will require continued adduct isolation studies.

  15. Syntheses of DL-[2-13C]leucine and its use in the preparation of [3-DL-[2-13C]leucine]oxytocin and [8-DL-[2-13C]leucine]oxytocin

    International Nuclear Information System (INIS)

    Viswanatha, V.; Larsen, B.; Hruby, V.J.

    1979-01-01

    DL-[2- 13 C]Leucine was prepared by condensing the sodium salt of ethyl acetamido-[2- 13 C]cyanoacetate with isobutylbromide in hexamethylphosphoroustriamide followed by acid hydrolysis. N-BOC-DL-[2- 13 C]Leucine was prepared and incorporated into [8-DL-[2- 13 C]leucine]oxytocin by total synthesis. The 13 C-labeled hormone derivative [8-[2- 13 C]leucine]oxytocin was separated from its 8-position diastereoisomer by partition chromatography. The specifically 13 C-labeled peptide hormone diastereoisomeric analog [3-DL-[2- 13 C]leucine]oxytocin also was prepared by solid phase peptide synthesis. No suitable solvent system for partition chromatography separation of the latter diastereoisomeric peptide mixture could be found. However an excellent preparative separation of the diastereoisomers could be obtained by reverse phase high pressure liquid chromatography on a partisil 10 M9 ODS column using the solvent system 0.05 M ammonium acetate (pH 4.0), acetonitrile (81:19, v/v) to give pure [3-[2- 13 C]leucine]oxytocin and [3-D-[2- 13 C]leucine]oxytocin. An excellent separation of [8-[2- 13 C]leucine]oxytocin and the corresponding delata-D-leucine diastereoisomer derivative could also be accomplished by high pressure liquid chromatography. (author)

  16. Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities.

    Science.gov (United States)

    Xie, Ran; Hong, Senlian; Chen, Xing

    2013-10-01

    Metabolic labeling of biomolecules with bioorthogonal functionalities enables visualization, enrichment, and analysis of the biomolecules of interest in their physiological environments. This versatile strategy has found utility in probing various classes of biomolecules in a broad range of biological processes. On the other hand, metabolic labeling is nonselective with respect to cell type, which imposes limitations for studies performed in complex biological systems. Herein, we review the recent methodological developments aiming to endow metabolic labeling strategies with cell-type selectivity. The cell-selective metabolic labeling strategies have emerged from protein and glycan labeling. We envision that these strategies can be readily extended to labeling of other classes of biomolecules. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Comparison of Glutamate Turnover in Nerve Terminals and Brain Tissue During [1,6-13C2]Glucose Metabolism in Anesthetized Rats.

    Science.gov (United States)

    Patel, Anant B; Lai, James C K; Chowdhury, Golam I M; Rothman, Douglas L; Behar, Kevin L

    2017-01-01

    The 13 C turnover of neurotransmitter amino acids (glutamate, GABA and aspartate) were determined from extracts of forebrain nerve terminals and brain homogenate, and fronto-parietal cortex from anesthetized rats undergoing timed infusions of [1,6- 13 C 2 ]glucose or [2- 13 C]acetate. Nerve terminal 13 C fractional labeling of glutamate and aspartate was lower than those in whole cortical tissue at all times measured (up to 120 min), suggesting either the presence of a constant dilution flux from an unlabeled substrate or an unlabeled (effectively non-communicating on the measurement timescale) glutamate pool in the nerve terminals. Half times of 13 C labeling from [1,6- 13 C 2 ]glucose, as estimated by least squares exponential fitting to the time course data, were longer for nerve terminals (Glu C4 , 21.8 min; GABA C2 21.0 min) compared to cortical tissue (Glu C4 , 12.4 min; GABA C2 , 14.5 min), except for Asp C3 , which was similar (26.5 vs. 27.0 min). The slower turnover of glutamate in the nerve terminals (but not GABA) compared to the cortex may reflect selective effects of anesthesia on activity-dependent glucose use, which might be more pronounced in the terminals. The 13 C labeling ratio for glutamate-C4 from [2- 13 C]acetate over that of 13 C-glucose was twice as large in nerve terminals compared to cortex, suggesting that astroglial glutamine under the 13 C glucose infusion was the likely source of much of the nerve terminal dilution. The net replenishment of most of the nerve terminal amino acid pools occurs directly via trafficking of astroglial glutamine.

  18. Optoacoustic 13C-breath test analyzer

    Science.gov (United States)

    Harde, Hermann; Helmrich, Günther; Wolff, Marcus

    2010-02-01

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

  19. Correlation between the 12C+12C, 12C+13C, and 13C+13C fusion cross sections

    Science.gov (United States)

    Notani, M.; Esbensen, H.; Fang, X.; Bucher, B.; Davies, P.; Jiang, C. L.; Lamm, L.; Lin, C. J.; Ma, C.; Martin, E.; Rehm, K. E.; Tan, W. P.; Thomas, S.; Tang, X. D.; Brown, E.

    2012-01-01

    The fusion cross section for 12C+13C has been measured down to Ec.m.=2.6 MeV, at which the cross section is of the order of 20 nb. By comparing the cross sections for the three carbon isotope systems, 12C+12C, 12C+13C, and 13C+13C, it is found that the cross sections for 12C+13C and 13C+13C provide an upper limit for the fusion cross section of 12C+12C over a wide energy range. After calibrating the effective nuclear potential for 12C+12C using the 12C+13C and 13C+13C fusion cross sections, it is found that a coupled-channels calculation with the ingoing wave boundary condition (IWBC) is capable of predicting the major peak cross sections in 12C+12C. A qualitative explanation for this upper limit is provided by the Nogami-Imanishi model and by level density differences among the compound nuclei. It is found that the strong resonance found at 2.14 MeV in 12C+12C exceeds this upper limit by a factor of more than 20. The preliminary result from the most recent measurement shows a much smaller cross section at this energy, which agrees with our predicted upper limit.

  20. Combination of liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry with 13C-labeling for chemical assignment of sulfur-containing metabolites in onion bulbs.

    Science.gov (United States)

    Nakabayashi, Ryo; Sawada, Yuji; Yamada, Yutaka; Suzuki, Makoto; Hirai, Masami Yokota; Sakurai, Tetsuya; Saito, Kazuki

    2013-02-05

    Phytochemicals containing heteroatoms (N, O, S, and halogens) often have biological activities that are beneficial to humans. Although targeted profiling methods for such phytochemicals are expected to contribute to rapid chemical assignments, thus making phytochemical genomics and crop breeding much more efficient, there are few profiling methods for the metabolites. Here, as an ultrahigh performance approach, we propose a practical profiling method for S-containing metabolites (S-omics) using onions (Allium cepa) as a representative species and (12)C- and (13)C-based mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses by liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC-FTICR-MS). Use of the ultrahigh quality data from FTICR-MS enabled simplifying the previous methods to determine specific elemental compositions. MS analysis with a resolution of >250,000 full width at half-maximum and a mass accuracy of ions from other ions on the basis of the natural abundance of (32)S and (34)S and the mass differences among the S isotopes. Comprehensive peak picking using the theoretical mass difference (1.99579 Da) between (32)S-containing monoisotopic ions and their (34)S-substituted counterparts led to the assignment of 67 S-containing monoisotopic ions from the (12)C-based MS spectra, which contained 4693 chromatographic ions. The unambiguous elemental composition of 22 ions was identified through comparative analysis of the (12)C- and (13)C-based MS spectra. Finally, of these, six ions were found to be derived from S-alk(en)ylcysteine sulfoxides and glutathione derivatives. This S-atom-driven approach afforded an efficient chemical assignment of S-containing metabolites, suggesting its potential application for screening not only S but also other heteroatom-containing metabolites in MS-based metabolomics.

  1. A systematic analysis of backbone amide assignments achieved via combinatorial selective labelling of amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Jeremy Craven, C. [University of Sheffield, Department of Biotechnology and Molecular Biology (United Kingdom); Al-Owais, Moza; Parker, Martin J. [University of Leeds, Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology (United Kingdom)], E-mail: m.j.parker@leeds.ac.uk

    2007-06-15

    With the advent of high-yield cell-free expressions systems, many researchers are exploiting selective isotope labelling of amino acids to increase the efficiency and accuracy of the NMR assignment process. We developed recently a combinatorial selective labelling (CSL) method capable of yielding large numbers of residue-type and sequence-specific backbone amide assignments, which involves comparing cross-peak intensities in {sup 1}H-{sup 15}N HSQC and 2D {sup 1}H-{sup 15}N HNCO spectra collected for five samples containing different combinations of {sup 13}C- and {sup 15}N-labelled amino acids [Parker MJ, Aulton-Jones M, Hounslow A, Craven C J (2004) J Am Chem Soc 126:5020-5021]. In this paper we develop a robust method for establishing the reliability of these assignments. We have performed a detailed statistical analysis of the CSL data collected for a model system (the B1 domain of protein G from Streptococcus), developing a scoring method which allows the confidence in assignments to be assessed, and which enables the effects of overlap on assignment fidelity to be predicted. To further test the scoring method and also to assess the performance of CSL in relation to sample quality, we have applied the method to the CSL data collected for GFP in our previous study.

  2. Detection and assignment of phosphoserine and phosphothreonine residues by {sup 13}C-{sup 31}P spin-echo difference NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    McIntosh, Lawrence P., E-mail: mcintosh@chem.ubc.ca; Kang, Hyun-Seo; Okon, Mark [University of British Columbia, Department of Biochemistry (Canada); Nelson, Mary L.; Graves, Barbara J. [University of Utah, Department of Oncological Sciences, Huntsman Cancer Institute (United States); Brutscher, Bernhard [CNRS, CEA, UJF, Institut de Biologie Structurale Jean-Pierre Ebel (France)], E-mail: bernhard.brutscher@ibs.fr

    2009-01-15

    A simple NMR method is presented for the identification and assignment of phosphorylated serine and threonine residues in {sup 13}C- or {sup 13}C/{sup 15}N-labeled proteins. By exploiting modest ({approx}5 Hz) 2- and 3-bond {sup 13}C-{sup 31}P scalar couplings, the aliphatic {sup 1}H-{sup 13}C signals from phosphoserines and phosphothreonines can be detected selectively in a {sup 31}P spin-echo difference constant time {sup 1}H-{sup 13}C HSQC spectrum. Inclusion of the same {sup 31}P spin-echo element within the {sup 13}C frequency editing period of an intraHNCA or HN(CO)CA experiment allows identification of the amide {sup 1}H{sup N} and {sup 15}N signals of residues (i) for which {sup 13}C{sup {alpha}}(i) or {sup 13}C{sup {alpha}}(i - 1), respectively, are coupled to a phosphate. Furthermore, {sup 31}P resonance assignments can be obtained by applying selective low power cw {sup 31}P decoupling during the spin-echo period. The approach is demonstrated using a PNT domain containing fragment of the transcription factor Ets-1, phosphorylated in vitro at Thr38 and Ser41 with the MAP kinase ERK2.

  3. 13C nuclear magnetic resonance data of lanosterol derivatives—Profiling the steric topology of the steroid skeleton via substituent effects on its 13C NMR

    Science.gov (United States)

    Dias, Jerry Ray; Gao, Hongwu

    2009-12-01

    The 13C NMR spectra of over 24 tetracyclic triterpenoid derivatives have been structurally analyzed. The 13C NMR chemical shifts allow one to probe the steric topology of the rigid steroid skeleton and inductive effects of its substituents. Use of deuterium labeling in chemical shift assignment and B-ring aromatic terpenoids are also featured.

  4. Accurate Determination of Leucine and Valine Side-chain Conformations using U-[{sup 15}N/{sup 13}C/{sup 2}H]/[{sup 1}H-(methine/methyl)-Leu/Val] Isotope Labeling, NOE Pattern Recognition, and Methine C{gamma}-H{gamma}/C{beta}-H{beta} Residual Dipolar Couplings

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Chun; Iwahara, Junji; Clore, G. Marius [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)], E-mail: mariusc@intra.niddk.nih.gov

    2005-10-15

    An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a {sup 15}N/{sup 13}C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of H{gamma}(i)-H{sub N}(i) and H{gamma}(i)-H{sub N}(i+1) NOEs in a 3D {sup 15}N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine {sup 13}C-{sup 1}H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA{sup Chitobiose}, a 34 kDa homotrimeric phosphotransferase protein.

  5. Selection of well labelled insulin fractions for radioimmunoassay use

    Energy Technology Data Exchange (ETDEWEB)

    Awh, O D; Kim, J R [Korea Atomic Energy Research Inst., Seoul (Republic of Korea)

    1980-06-01

    Selection methods of well labelled insulin fractions based on two different criteria were compared to establish an efficient low level RIA of insulin and to elucidate the correlation between the immunoreactivity and the charcoal-adsorptivity of the radioiodine labelled insulin. The result indicated that the selection of well labelled insulin fractions by means of a charcoal-adsorption test is inappropriate. Generally, the distribution of radioactivity, antibody-bindability, and charcoal-adsorptivity of the labelled insulin was not consistent with each other. Thus, the selection should be carried out for every labelling batch to get the utmost assay reliability by antibody-bindability but not by charcoal-adsorptivity. By using the well selected labelled insulin fractions based on antibody-binding, a correct assay for a reference serum was possible, and by extending the incubation time up to 96 hrs, a sharp dose response curve could be obtained even in the range of below 5 ..mu..U/ml standard insulin doses.

  6. Assessment of Hepatic Mitochondrial Oxidation and Pyruvate Cycling in NAFLD by (13)C Magnetic Resonance Spectroscopy

    DEFF Research Database (Denmark)

    Petersen, Kitt Mia Falck; Befroy, Douglas E; Dufour, Sylvie

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and there is great interest in understanding the potential role of alterations in mitochondrial metabolism in its pathogenesis. To address this question, we assessed rates of hepatic mitochondrial oxidation...... in subjects with and without NAFLD by monitoring the rate of (13)C labeling in hepatic [5-(13)C]glutamate and [1-(13)C]glutamate by (13)C MRS during an infusion of [1-(13)C]acetate. We found that rates of hepatic mitochondrial oxidation were similar between NAFLD and control subjects. We also assessed rates...

  7. In Folio Respiratory Fluxomics Revealed by {sup 13}C Isotopic Labeling and H/D Isotope Effects Highlight the Non-cyclic Nature of the Tricarboxylic Acid 'Cycle' in Illuminated Leaves

    Energy Technology Data Exchange (ETDEWEB)

    Tcherkez, G.; Mahe, A.; Gauthier, P.; Hodges, M. [Institut de Biotechnologie des Plantes, Plateforme Metabolisme-Metabolome IFR87, Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Tcherkez, G.; Mauve, C.; Cornic, G. [Laboratoire d' Ecophysiologie Vegetale, Ecologie Systematique Evolution (G.C.), Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Gout, E.; Bligny, R. [Laboratoire de Physiologie Cellulaire Vegetale, Commissariat a l' Energie Atomique-Grenoble, 38054 Grenoble cedex 9 (France)

    2009-07-01

    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, {sup 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. (authors)

  8. In Folio Respiratory Fluxomics Revealed by 13C Isotopic Labeling and H/D Isotope Effects Highlight the Non-cyclic Nature of the Tricarboxylic Acid 'Cycle' in Illuminated Leaves

    International Nuclear Information System (INIS)

    Tcherkez, G.; Mahe, A.; Gauthier, P.; Hodges, M.; Tcherkez, G.; Mauve, C.; Cornic, G.; Gout, E.; Bligny, R.

    2009-01-01

    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. (authors)

  9. In Folio Respiratory Fluxomics Revealed by {sup 13}C Isotopic Labeling and H/D Isotope Effects Highlight the Non-cyclic Nature of the Tricarboxylic Acid 'Cycle' in Illuminated Leaves

    Energy Technology Data Exchange (ETDEWEB)

    Tcherkez, G; Mahe, A; Gauthier, P; Hodges, M [Institut de Biotechnologie des Plantes, Plateforme Metabolisme-Metabolome IFR87, Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Tcherkez, G; Mauve, C; Cornic, G [Laboratoire d' Ecophysiologie Vegetale, Ecologie Systematique Evolution (G.C.), Batiment 630, Universite Paris-Sud 11, 91405 Orsay cedex (France); Gout, E; Bligny, R [Laboratoire de Physiologie Cellulaire Vegetale, Commissariat a l' Energie Atomique-Grenoble, 38054 Grenoble cedex 9 (France)

    2009-07-01

    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, {sup 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. (authors)

  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 Leaves1[W

    Science.gov (United States)

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

    2009-01-01

    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, 13C 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. Evaluating Varied Label Designs for Use with Medical Devices: Optimized Labels Outperform Existing Labels in the Correct Selection of Devices and Time to Select.

    Directory of Open Access Journals (Sweden)

    Laura Bix

    Full Text Available Effective standardization of medical device labels requires objective study of varied designs. Insufficient empirical evidence exists regarding how practitioners utilize and view labeling.Measure the effect of graphic elements (boxing information, grouping information, symbol use and color-coding to optimize a label for comparison with those typical of commercial medical devices.Participants viewed 54 trials on a computer screen. Trials were comprised of two labels that were identical with regard to graphics, but differed in one aspect of information (e.g., one had latex, the other did not. Participants were instructed to select the label along a given criteria (e.g., latex containing as quickly as possible. Dependent variables were binary (correct selection and continuous (time to correct selection.Eighty-nine healthcare professionals were recruited at Association of Surgical Technologists (AST conferences, and using a targeted e-mail of AST members.Symbol presence, color coding and grouping critical pieces of information all significantly improved selection rates and sped time to correct selection (α = 0.05. Conversely, when critical information was graphically boxed, probability of correct selection and time to selection were impaired (α = 0.05. Subsequently, responses from trials containing optimal treatments (color coded, critical information grouped with symbols were compared to two labels created based on a review of those commercially available. Optimal labels yielded a significant positive benefit regarding the probability of correct choice ((P<0.0001 LSM; UCL, LCL: 97.3%; 98.4%, 95.5%, as compared to the two labels we created based on commercial designs (92.0%; 94.7%, 87.9% and 89.8%; 93.0%, 85.3% and time to selection.Our study provides data regarding design factors, namely: color coding, symbol use and grouping of critical information that can be used to significantly enhance the performance of medical device labels.

  12. Contribution of exogenous substrates to acetyl coenzyme A: Measurement by 13C NMR under non-steady-state conditions

    International Nuclear Information System (INIS)

    Malloy, C.R.; Jeffrey, F.M.H.; Thompson, J.R.; Sherry, A.D.

    1990-01-01

    A method is presented for the rapid determination of substrate selection in a manner that is not restricted to conditions of metabolic and isotopic steady state. Competition between several substrates can be assessed directly and continuously in a single experiment, allowing the effect of interventions to be studied. It is shown that a single proton-decoupled 13 C NMR spectrum of glutamate provides a direct measure of the contribution of exogenous 13 C-labeled substrates to acetyl-CoA without measurement of oxygen consumption and that steady-state conditions need not apply. Two sets of experiments were performed: one in which a metabolic steady state but a non-steady-state 13 C distribution was achieved and another in which both metabolism and labeling were not at steady state. In the first group, isolated rat hearts were supplied with [1,2- 13 C]acetate, [3- 13 C]lactate, and unlabeled glucose. 13 C NMR spectra of extracts from hearts perfused under identical conditions for 5 or 30 min were compared. In spite of significant differences in the spectra, the measured contributions of acetate, lactate, and unlabeled sources to acetyl-CoA were the same. In the second set of experiments, the same group of labeled substrates was used in a regional ischemia model in isolated rabbit hearts to show regional differences in substrate utilization under both metabolic and isotopic non steady state. The time resolution of these measurements may not be limited by technical contraints but by the rate of carbon flux in the citric acid cycle. Although this technique is demonstrated for the heart, it is applicable to all tissues

  13. Catalytic dehydration of ethanol for poly 13 C compounds synthesis

    International Nuclear Information System (INIS)

    Almasan, Valer; Marginean, Petru; Lazar, Mihaela; Tusa, Florina

    2003-01-01

    Classical methods for the synthesis of organic compounds are not very well applied in the case of 13 C labeled compounds. One of the principal demands is to find the best method to transform a small quantity of isotopic reagent with a very high yield. In this case to obtain 13 C 2 chloroethanol from 13 C 2 ethanol there are two synthesis steps: - catalytic dehydration of ethanol to ethylene; - ethylene double bounding saturation: either via ethylene oxide (30% yield) or in diluted solution of chlorine. For the first step of synthesis we choose the thermal dehydration over alumina catalyst at 400 deg C. There were tested 2 samples of g alumina with 255 m 2 /g and 355 m 2 /g with very good results. In the second step of the synthesis we used the chlorine addition to ethylene in very diluted water solution. We have built a reactor which combined the two steps of this synthesis method to produce 13 C 2 chloroethanol from 13 C 2 ethanol. The global yield of method was 42%. (authors)

  14. Tritium labelling of testosteron by selective hydrogenation of dihydrotestosteron

    International Nuclear Information System (INIS)

    Postolache, Cristian; Matei, Lidia; Simion, Elena; Barna, Catalina; Condac, Eduard

    2002-01-01

    Elemental tritium is obtained during the decontamination process of the moderator from Cernavoda Nuclear Power Plant. It might be stocked for use in controlled fusion, in a relatively far future, or, it might be immediately used as raw material in the synthesis of labelled compounds with important economic value. Labelling of testosteron with tritium was necessary for the carrying out of radiometric and molecular biology studies concerning androgen dependent diseases. Testosteron was labelled by selective hydrogenation of 1,2 dihydrotestosteron acetate. The forerunner was synthesized in two steps: 1) esterification of testosteron using acetic anhydride, and 2) selective dehydrogenation with 2,6-dichloro-3,5-dicyan-1,4 quinone (DDQ) of the ester formed in the first step. Testosteron acetate was synthesized and purified with yields of 73%, and 80%, respectively. The dehydrogenation process was characterized by yields of 82% for synthesis and 33% for purification. The tritium labelled hormone was obtained in two steps: 1) selective hydrogenation of Δ 1 - testosteron acetate in the presence of T 2 gas, at low pressure, and 2) hydrolysis of the ester at basic pH. The raw product obtained was purified by preparative thin layer chromatography. The physical and chemical characterization of labelled testosteron reveals a radiochemical purity higher than 98% and a specific activity of 53.4 Ci/mmol. (authors)

  15. In vivo {sup 13}C MRS studies of carbohydrate metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Halliday, Jane

    2003-07-01

    The work described in this thesis was performed by the author, except where indicated, within the Magnetic Resonance Centre at the University of Nottingham during the period between October 1999 and October 2002. Although much is known about the major pathways of carbohydrate metabolism, there is still much to be learnt about the exact mechanisms of many of these pathways. Of particular interest is how these pathways are modified under different physiological conditions and in diseased states. {sup 13}C NMR spectroscopy provides a non-invasive means for studying carbohydrate metabolism in vivo, and the work presented within this thesis gives two such examples of this in human subjects. Natural abundance {sup 13}C NMR spectroscopy was used to measure glycogen levels in gastrocnemius muscle. The diurnal changes in response to mixed meals were measured in both type 2 diabetic subjects and age and weight matched controls. Metabolic studies were performed to complement the NMR measurements. The data obtained in these studies show the effect of the failure of muscle glucose storage upon post-prandial hyperglycaemia despite a supra-normal increase in plasma insulin in type 2 diabetes. {sup 13}C NMR spectroscopy was also used to study cerebral metabolism. Accumulation of {sup 13}C label into glutamate and glutamine following infusion of [1{sup 13}C] glucose allows the determination of the rates of the TCA cycle (F{sub TCA}) and neurotransmitter cycling (F{sub cyc}). These rates were measured in the visual cortex under control and activated conditions. The increases seen in F{sub TCA} upon activation, together with the lack of label accumulation in lactate, suggest that cerebral glucose metabolism is oxidative, even during strong activation. No conclusion can be made as to whether or not a similar increase is seen in F{sub cyc} due to the large associated errors in these values. (author)

  16. 1H, 15N and 13C NMR Assignments of Mouse Methionine Sulfoxide Reductase B2

    Science.gov (United States)

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

    2011-01-01

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

  17. Intracellular PHB conversion in a type II methanotroph studied by 13 C NMR

    NARCIS (Netherlands)

    Vecherskaya, M.; Dijkema, C.; Stams, A.J.M.

    2001-01-01

    Poly-g-hydroxybutyrate (PHB) formation under aerobic conditions via incorporation of [13C-2]acetate as a cosubstrate and its intracellular degradation under anaerobic conditions in a Type II methanotroph was studied by 13C NMR. During PHB synthesis in the presence of labelled acetate, low levels of

  18. Label-free and selective nonlinear fiber-optical biosensing

    DEFF Research Database (Denmark)

    Ott, Johan Raunkjær; Heuck, Mikkel; Agger, Christian

    2008-01-01

    We demonstrate that the inherent nonlinearity of a microstructured optical fiber (MOF) may be used to achieve label-free selective biosensing, thereby eliminating the need for post-processing of the fiber. This first nonlinear biosensor utilizes a change in the modulational instability (MI) gain...... for optimizing the sensitivity. The nonlinear sensor shows a sensitivity of around 10.4nm/nm, defined as the shift in resonance wavelength per nm biolayer, which is a factor of 7.5 higher than the hitherto only demonstrated label-free MOF biosensor....

  19. Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose

    Science.gov (United States)

    Moran, Nancy E.; Rogers, Randy B.; Lu, Chi-Hua; Conlon, Lauren E.; Lila, Mary Ann; Clinton, Steven K.; Erdman, John W.

    2013-01-01

    While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched 13C-lycopene for human bioavailability and metabolism studies. To enhance the 13C-enrichment and yields of labeled lycopene from the hp-1 tomato cell line, cultures were first grown in 13C-glucose media for three serial batches and produced increasing proportions of uniformly labeled lycopene (14.3 +/− 1.2 %, 39.6 +/− 0.5 %, and 48.9 +/− 1.5% with consistent yields (from 5.8 to 9 mg/L). An optimized 9-day-long 13C-loading and 18-day-long labeling strategy developed based on glucose utilization and lycopene yields, yielded 13C-lycopene with 93% 13C isotopic purity, and 55% of isotopomers were uniformly labeled. Furthermore, an optimized acetone and hexane extraction led to a four-fold increase in lycopene recovery from cultures compared to a standard extraction. PMID:23561155

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

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Rodolfo A. (Santa Fe, NM), Unkefer; Clifford J. (Los Alamos, NM), Alvarez; Marc, A [Santa Fe, NM

    2012-06-12

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

  1. Electric dipole moment of 13C

    Science.gov (United States)

    Yamanaka, Nodoka; Yamada, Taiichi; Hiyama, Emiko; Funaki, Yasuro

    2017-06-01

    We calculate for the first time the electric dipole moment (EDM) of 13C generated by the isovector charge conjugation-parity (CP)-odd pion exchange nuclear force in the α -cluster model, which describes well the structures of low-lying states of the 13C nucleus. The linear dependence of the EDM of 13C on the neutron EDM and the isovector CP-odd nuclear coupling is found to be d13C=-0.33 dn-0.0020 G¯π(1 ) . The linear enhancement factor of the CP-odd nuclear coupling is smaller than that of the deuteron, due to the difference of the structure between the 1 /21- state and the opposite-parity (1 /2+ ) states. We clarify the role of the structure played in the enhancement of the EDM. This result provides good guiding principles to search for other nuclei with large enhancement factor. We also mention the role of the EDM of 13C in determining the new physics beyond the standard model.

  2. Evaluation of carbon transfers in cattle and humans using 13C

    International Nuclear Information System (INIS)

    Masuda, Tsuyoshi; Tako, Yasuhiro; Nakamura, Yuji

    2010-01-01

    In the safety assessment made around the spent nuclear fuel reprocessing plant in Rokkasho, Aomori, among radioactive nuclides released from the plant, 14 C is expected to be the largest contributor to radiation dose received by the neighboring population through agricultural and dairy products. The objectives of this study are to clarify the transfer of 14 C from grass to beef and milk and its metabolism in the human body experimentally. (1) 13 C-labeled grass was fed for 28 days to beef cattle and cows. 13 C isotopic ratio was measured in serum and other samples including muscle of beef cattle and milk of dairy cow. The 13 C rations in milk, breath air, urine and feces decreased very rapidly within 3 day after cessation of the administration of 13 C-labeled feed. However, a slow decrease in 13 C ratio was observed in muscle and serum. (2) 13 C isotopic ratios were measured in breath air, urine, feces and serum over 16 weeks in humans who were orally administered of 13 C labeled leucine, palmitic acid, glucose, boiled rice and soymilk, respectively. Residual 13 C in their bodies experimentally observed were lower than the estimates by the ICRP metabolic model for organic carbon ingestion. (author)

  3. Selective detection of carbon-13, nitrogen-15, and deuterium labeled metabolites by capillary gas chromatography-chemical reaction interface/mass spectrometry

    International Nuclear Information System (INIS)

    Chace, D.H.; Abramson, F.P.

    1989-01-01

    We have applied a new chemical reaction interface/mass spectrometer technique (CRIMS) to the selective detection of 13C-, 15N-, and 2H-labeled phenytoin and its metabolites in urine following separation by capillary gas chromatography. The microwave-powered chemical reaction interface converts materials from their original forms into small molecules whose mass spectra serve to identify and quantify the nuclides that make up each analyte. The presence of each element is followed by monitoring the isotopic variants of CO2, NO, or H2 that are produced by the chemical reaction interface. Chromatograms showing only enriched 13C and 15N were produced by subtracting the abundance of naturally occurring isotopes from the observed M + 1 signal. A selective chromatogram of 2H (D) was obtained by measuring HD at m/z 3.0219 with a resolution of 2000. Metabolites representing less than 1.5% of the total labeled compounds could be identified in the chromatogram. Detection limits from urine of 380 pg/mL of a 15N-labeled metabolite, 7 ng/mL of a 13C-labeled metabolite, and 16 ng/mL of a deuterium labeled metabolite were determined at a signal to noise ratio of 2. Depending on the isotope examined, a linear dynamic range of 250-1000 was observed using CRIMS. To identify many of these labeled peaks (metabolites), the chromatographic analysis was repeated with the chemical reaction interface turned off and mass spectra obtained at the retention times found in the CRIMS experiment. CRIMS is a new analytical method that appears to be particularly useful for metabolism studies

  4. In vivo 13C MRS studies of carbohydrate metabolism

    International Nuclear Information System (INIS)

    Halliday, Jane

    2003-01-01

    The work described in this thesis was performed by the author, except where indicated, within the Magnetic Resonance Centre at the University of Nottingham during the period between October 1999 and October 2002. Although much is known about the major pathways of carbohydrate metabolism, there is still much to be learnt about the exact mechanisms of many of these pathways. Of particular interest is how these pathways are modified under different physiological conditions and in diseased states. 13 C NMR spectroscopy provides a non-invasive means for studying carbohydrate metabolism in vivo, and the work presented within this thesis gives two such examples of this in human subjects. Natural abundance 13 C NMR spectroscopy was used to measure glycogen levels in gastrocnemius muscle. The diurnal changes in response to mixed meals were measured in both type 2 diabetic subjects and age and weight matched controls. Metabolic studies were performed to complement the NMR measurements. The data obtained in these studies show the effect of the failure of muscle glucose storage upon post-prandial hyperglycaemia despite a supra-normal increase in plasma insulin in type 2 diabetes. 13 C NMR spectroscopy was also used to study cerebral metabolism. Accumulation of 13 C label into glutamate and glutamine following infusion of [1 1 3 C] glucose allows the determination of the rates of the TCA cycle (F TCA ) and neurotransmitter cycling (F cyc ). These rates were measured in the visual cortex under control and activated conditions. The increases seen in F TCA upon activation, together with the lack of label accumulation in lactate, suggest that cerebral glucose metabolism is oxidative, even during strong activation. No conclusion can be made as to whether or not a similar increase is seen in F cyc due to the large associated errors in these values. (author)

  5. Metabolic Characterization of Acutely Isolated Hippocampal and Cerebral Cortical Slices Using [U-13C]Glucose and [1,2-13C]Acetate as Substrates.

    Science.gov (United States)

    McNair, Laura F; Kornfelt, Rasmus; Walls, Anne B; Andersen, Jens V; Aldana, Blanca I; Nissen, Jakob D; Schousboe, Arne; Waagepetersen, Helle S

    2017-03-01

    Brain slice preparations from rats, mice and guinea pigs have served as important tools for studies of neurotransmission and metabolism. While hippocampal slices routinely have been used for electrophysiology studies, metabolic processes have mostly been studied in cerebral cortical slices. Few comparative characterization studies exist for acute hippocampal and cerebral cortical slices, hence, the aim of the current study was to characterize and compare glucose and acetate metabolism in these slice preparations in a newly established incubation design. Cerebral cortical and hippocampal slices prepared from 16 to 18-week-old mice were incubated for 15-90 min with unlabeled glucose in combination with [U- 13 C]glucose or [1,2- 13 C]acetate. Our newly developed incubation apparatus allows accurate control of temperature and is designed to avoid evaporation of the incubation medium. Subsequent to incubation, slices were extracted and extracts analyzed for 13 C-labeling (%) and total amino acid contents (µmol/mg protein) using gas chromatography-mass spectrometry and high performance liquid chromatography, respectively. Release of lactate from the slices was quantified by analysis of the incubation media. Based on the measured 13 C-labeling (%), total amino acid contents and relative activity of metabolic enzymes/pathways, we conclude that the slice preparations in the current incubation apparatus exhibited a high degree of metabolic integrity. Comparison of 13 C-labeling observed with [U- 13 C]glucose in slices from cerebral cortex and hippocampus revealed no significant regional differences regarding glycolytic or total TCA cycle activities. On the contrary, results from the incubations with [1,2- 13 C]acetate suggest a higher capacity of the astrocytic TCA cycle in hippocampus compared to cerebral cortex. Finally, we propose a new approach for assessing compartmentation of metabolite pools between astrocytes and neurons using 13 C-labeling (%) data obtained from

  6. (13)C MR spectroscopy study of lactate as substrate for rat brain.

    Science.gov (United States)

    Qu, H; Håberg, A; Haraldseth, O; Unsgård, G; Sonnewald, U

    2000-01-01

    In order to address the question whether lactate in blood can serve as a precursor for cerebral metabolites, fully awake rats were injected intravenously with [U-(13)C]lactate or [U-(13)C]glucose followed 15 min later by decapitation. Incorporation of label from [U-(13)C]glucose was seen mainly in glutamate, GABA, glutamine, aspartate, alanine and lactate. More label was found in glutamate than glutamine, underscoring the predominantly neuronal metabolism of pyruvate from [U-(13)C]glucose. It was estimated that the neuronal metabolism of acetyl CoA from glucose accounts for at least 66% and the glial for no more than 34% of the total glucose consumption. When [U-(13)C]lactate was the precursor, label incorporation was similar to that observed from [U-(13)C]glucose, but much reduced. Plasma analysis revealed the presence of approximately equal amounts of [1,2,3-(13)C]- and [1,2-(13)C]glucose, showing gluconeogenesis from [U-(13)C]lactate. It was thus possible that the labeling seen in the cerebral amino acids originated from labeled glucose, not [U-(13)C]lactate. However, the presence of significantly more label in [U-(13)C]- than in [2,3-(13)C]alanine demonstrated that [U-(13)C]lactate did indeed cross the blood-brain barrier, and was metabolized further in the brain. Furthermore, contributions from pyruvate carboxylase (glial enzyme) were detectable in glutamine, glutamate and GABA, and were comparatively more pronounced in the glucose group. This indicated that relatively more pyruvate from lactate than glucose was metabolized in neurons. Surprisingly, the same amount of lactate was synthesized via the tricarboxylic acid cycle in both groups, indicating transfer of neurotransmitters from the neuronal to the astrocytic compartment, as previous studies have shown that this lactate is synthesized primarily in astrocytes. Taking into consideration that astrocytes take up glutamate more avidly than GABA, it is conceivable that neuronal lactate metabolism was more

  7. Multi-Label Learning via Random Label Selection for Protein Subcellular Multi-Locations Prediction.

    Science.gov (United States)

    Wang, Xiao; Li, Guo-Zheng

    2013-03-12

    Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multi-location proteins to multiple proteins with single location, which doesn't take correlations among different subcellular locations into account. In this paper, a novel method named RALS (multi-label learning via RAndom Label Selection), is proposed to learn from multi-location proteins in an effective and efficient way. Through five-fold cross validation test on a benchmark dataset, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark datasets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multi-locations of proteins. The prediction web server is available at http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage.

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

    Directory of Open Access Journals (Sweden)

    Marin de Mas Igor

    2011-10-01

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

  9. {sup 13}C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Mitsuhiro [Kumamoto University, Department of Structural BioImaging, Faculty of Life Sciences (Japan); Miyanoiri, Yohei [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Terauchi, Tsutomu [Tokyo Metropolitan University, Graduate School of Science and Engineering (Japan); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2016-09-15

    Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14–Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D {sup 13}C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically {sup 13}C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14–Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m{sub c14} and m{sub c38}). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for {sup 13}C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m{sub c38} isomerization, the {sup 1}H-{sup 13}C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.

  10. 13C separation by IRMPD of halogenated difluoromethanes

    International Nuclear Information System (INIS)

    Ma Peihua; Chen Guancheng; Wu Bin; Liu Julin; Jing Yan; Chu Minxiong; Arai, Shigeyoshi.

    1995-01-01

    Isotopically-selective consecutive two-stage infrared multiphoton dissociation (IRMPD) of halogenated difluoromethanes in the presence of scavengers produces carbon-13 over 95 %. The reaction mechanism for the IRMPD of mixture of CHClF 2 and HI can be explained by a series of first-order dissociation reactions and followed radical-scavenger reactions occurred in a continuous irradiation procedure. Furthermore, 13 C enrichment at laboratory scaling-up level by the 13 C selective IRMPD of CHClF 2 /Br 2 mixture has been investigated in a flow reactor. The 13 C production rates, 13 C atomic fractions in the CBr 2 F 2 products and 13 C depletions in the CHClF 2 reactants at different flow rates and laser repetition frequencies were examined to optimize the parameters suitable for large-scale production of carbon isotope. The data obtained from the flow tests demonstrated a 40 mg h -1 production rate for CB 2 F 2 at 65 % carbon-13 by using a 40 W (4J, 10 Hz) laser beam focused with a lens of focal length 120 cm. If a reliable TEA CO 2 laser can be operated with 100 W (10 J, 10 Hz) output, the production rate of CBr 2 F 2 for carbon-13 at 60 % can attain 200 mg h -1 . The measurements of spatial profile of focused laser beam imply a 2 g h -1 production rate for the 60 % carbon-13 product for an incident power of 200 W (20 J, 10 Hz). (author)

  11. Alpha Resonant States in 13C

    International Nuclear Information System (INIS)

    Rodrigues, M. R. D.; Borello-Lewin, T.; Horodynski-Matsushigue, L. B.; Duarte, J. L. M.; Rodrigues, C. L.; Souza, M. A.; Miyake, H.; Cunsolo, A.; Cappuzzello, F.; Ukita, G. M.

    2011-01-01

    The 9 Be( 6 Li,d) 13 C reaction was used to investigate alpha resonant states in 13 C up to 15 MeV of excitation. The reaction was measured at a bombarding energy of 25.5 MeV employing the Sao Paulo Pelletron-Enge-Spectrograph facility and the nuclear emulsion detection technique. An energy resolution of 50 keV was obtained. Several narrow alpha resonant states not previously measured were detected, in particular the one at the (3α+n) threshold populated by an L = 2 transfer, revealing a 9 Be+α component for the 1/2 - cluster state candidate at this threshold. Experimental angular distributions are presented in comparison with DWBA predictions.

  12. Galactose oxidation using 13C in healthy and galactosemic children

    Directory of Open Access Journals (Sweden)

    D.R. Resende-Campanholi

    2015-03-01

    Full Text Available Galactosemia is an inborn error of galactose metabolism that occurs mainly as the outcome of galactose-1-phosphate uridyltransferase (GALT deficiency. The ability to assess galactose oxidation following administration of a galactose-labeled isotope (1-13C-galactose allows the determination of galactose metabolism in a practical manner. We aimed to assess the level of galactose oxidation in both healthy and galactosemic Brazilian children. Twenty-one healthy children and seven children with galactosemia ranging from 1 to 7 years of age were studied. A breath test was used to quantitate 13CO2 enrichment in exhaled air before and at 30, 60, and 120 min after the oral administration of 7 mg/kg of an aqueous solution of 1-13C-galactose to all children. The molar ratios of 13CO2 and 12CO2 were quantified by the mass/charge ratio (m/z of stable isotopes in each air sample by gas-isotope-ratio mass spectrometry. In sick children, the cumulative percentage of 13C from labeled galactose (CUMPCD in the exhaled air ranged from 0.03% at 30 min to 1.67% at 120 min. In contrast, healthy subjects showed a much broader range in CUMPCD, with values from 0.4% at 30 min to 5.58% at 120 min. The study found a significant difference in galactose oxidation between children with and without galactosemia, demonstrating that the breath test is useful in discriminating children with GALT deficiencies.

  13. Robust refocusing of 13C magnetization in multidimensional NMR experiments by adiabatic fast passage pulses

    International Nuclear Information System (INIS)

    Zweckstetter, Markus; Holak, Tad A.

    1999-01-01

    We show that adiabatic fast passage (AFP) pulses are robust refocusing elements of transverse 13 C magnetization in multidimensional NMR experiments. A pair of identical AFP pulses can refocus selected parts or a complete 13 C chemical shift range in 13 C spectra. In the constant time 13 C- 1 H HSQC, replacement of attenuated rectangular pulses by selective AFP pulses results in a sensitivity enhancement of up to a factor of 1.8. In the 3D CBCA(CO)NH the signal-to-noise ratio is increased by a factor of up to 1.6

  14. Inhibitors of the alpha-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neurons.

    Science.gov (United States)

    Santos, Sónia Sá; Gibson, Gary E; Cooper, Arthur J L; Denton, Travis T; Thompson, Charles M; Bunik, Victoria I; Alves, Paula M; Sonnewald, Ursula

    2006-02-15

    Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1-13C]glucose and [U-13C]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4-13C]glutamate from [1-13C]glucose, a reduction in label in glutamate derived from [1-13C]glucose/[U-13C]glutamate through a second turn of the TCA cycle and a decline in the amounts of gamma-aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U-13C]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased alpha-ketoglutarate formation; increased transamination of alpha-ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from alpha-ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases.

  15. Determination of urea 13C in urea 13C mixed powder by HPLC

    International Nuclear Information System (INIS)

    Zhong Jianguo; Song Tianqi

    2006-01-01

    A HPLC method is developed for determination of Urea 13 C in Urea 13 C Mixed Powder. A Alltech Econosphere NH2 column (250 mm x 4.6 mm, 5 μm)is used as stationary phrase, a mixture of V(acetonitrile): V(methanol): V(water) = 900 : 100: 10 is used as mobile phase and the flow rate is l mL·min -1 , UV detection wavelength is performed at 200 nm. The calibration curve shows good linearity in the range of 0.2-1.0 g·L -1 of Urea 13 C, y=2.548 x 10 6 x + 4.005 x 10 4 , r=0.9999, and the averaged recovery is 100.6%. The method is simple and accurate, and can be used for the quality control of Urea 13C Mixed Powder. (authors)

  16. Conditions to obtain precise and true measurements of the intramolecular {sup 13}C distribution in organic molecules by isotopic {sup 13}C nuclear magnetic resonance spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Bayle, Kevin [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France); Gilbert, Alexis [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Earth–Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8551 (Japan); Julien, Maxime [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France); Yamada, Keita [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Silvestre, Virginie; Robins, Richard J.; Akoka, Serge [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France); Yoshida, Naohiro [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Earth–Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8551 (Japan); Remaud, Gérald S., E-mail: gerald.remaud@univ-nantes.fr [EBSI Team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3 (France)

    2014-10-10

    Highlights: • Evaluation of the trueness and precision criteria of isotopic {sup 13}C NMR spectrometry. • Use of bi-labelled [1,2-{sup 13}C{sub 2}]acetic acid to determine the performance of the instrumental response. • Inter-calibration of the {sup 13}C intramolecular composition of acetic acid using the technique GC-Py–irm-MS. - Abstract: Intramolecular {sup 13}C composition gives access to new information on the (bio) synthetic history of a given molecule. Isotopic {sup 13}C NMR spectrometry provides a general tool for measuring the position-specific {sup 13}C content. As an emerging technique, some aspects of its performance are not yet fully delineated. This paper reports on (i) the conditions required to obtain satisfactory trueness and precision for the determination of the internal {sup 13}C distribution, and (ii) an approach to determining the “absolute” position-specific {sup 13}C content. In relation to (i), a precision of <1% can be obtained whatever the molecule on any spectrometer, once quantitative conditions are met, in particular appropriate proton decoupling efficiency. This performance is a prerequisite to the measurement of isotope fractionation either on the transformed or residual compound when a chemical reaction or process is being studied. The study of the trueness has revealed that the response of the spectrometer depends on the {sup 13}C frequency range of the studied molecule, i.e. the chemical shift range. The “absolute value” and, therefore, the trueness of the {sup 13}C NMR measurements has been assessed on acetic acid and by comparison to the results obtained on the fragments from COOH and CH{sub 3} by isotopic mass spectrometry coupled to a pyrolysis device (GC-Py–irm-MS), this technique being the reference method for acetic acid. Of the two NMR spectrometers used in this work, one gave values that corresponded to those obtained by GC-Py–irm-MS (thus, the “true” value) while the other showed a bias, which was

  17. Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed

    International Nuclear Information System (INIS)

    Raap, J.; Hollander, J.; Ovchinnikova, T. V.; Swischeva, N. V.; Skladnev, D.; Kiihne, S.

    2006-01-01

    Interactions between 15 N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortunately, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in 13 C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C α signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the 31 P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The 13 C-peptide peaks were selectively detected in a 13 C-detected 1 H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The 13 C-MAOSS results thus independently confirms previous findings from 15 N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution 13 C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and

  18. Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons.

    Science.gov (United States)

    Brekke, Eva M F; Walls, Anne B; Schousboe, Arne; Waagepetersen, Helle S; Sonnewald, Ursula

    2012-09-01

    The brain is highly susceptible to oxidative injury, and the pentose phosphate pathway (PPP) has been shown to be affected by pathological conditions, such as Alzheimer's disease and traumatic brain injury. While this pathway has been investigated in the intact brain and in astrocytes, little is known about the PPP in neurons. The activity of the PPP was quantified in cultured cerebral cortical and cerebellar neurons after incubation in the presence of [2-(13)C]glucose or [3-(13)C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism of (13)C-labeled glucose via the PPP does not appear to contribute to the production of releasable lactate, it contributes to labeling of tricarboxylic acid (TCA) cycle intermediates and related amino acids. Based on glutamate isotopomers, it was calculated that PPP activity accounts for ~6% of glucose metabolism in cortical neurons and ~4% in cerebellar neurons. This is the first demonstration that pyruvate generated from glucose via the PPP contributes to the synthesis of acetyl CoA for oxidation in the TCA cycle. Moreover, the fact that (13)C labeling from glucose is incorporated into glutamate proves that both the oxidative and the nonoxidative stages of the PPP are active in neurons.

  19. 6. Label-free selective plane illumination microscopy of tissue samples

    Directory of Open Access Journals (Sweden)

    Muteb Alharbi

    2017-10-01

    Conclusion: Overall this method meets the demands of the current needs for 3D imaging tissue samples in a label-free manner. Label-free Selective Plane Microscopy directly provides excellent information about the structure of the tissue samples. This work has highlighted the superiority of Label-free Selective Plane Microscopy to current approaches to label-free 3D imaging of tissue.

  20. Multi site Kinetic Modeling of 13C Metabolic MR Using [1-13C]Pyruvate

    International Nuclear Information System (INIS)

    Damian, P.A.G.; Sperl, J.I.; Janich, M.A.; Wiesinger, F.; Schulte, R.F.; Menzel, M.I.; Damian, P.A.G.; Damian, P.A.G.; Haase, A.; Janich, M.A.; Schwaiger, M.; Janich, M.A.; Khegai, O.; Glaser, S.J.

    2014-01-01

    Hyperpolarized 13 C imaging allows real-time in vivo measurements of metabolite levels. Quantification of metabolite conversion between [1- 13 C]pyruvate and downstream metabolites [1- 13 C]alanine, [1- 13 C]lactate, and [ 13 C] bicarbonate can be achieved through kinetic modeling. Since pyruvate interacts dynamically and simultaneously with its downstream metabolites, the purpose of this work is the determination of parameter values through a multi site, dynamic model involving possible biochemical pathways present in MR spectroscopy. Kinetic modeling parameters were determined by fitting the multi site model to time-domain dynamic metabolite data. The results for different pyruvate doses were compared with those of different two-site models to evaluate the hypothesis that for identical data the uncertainty of a model and the signal-to-noise ratio determine the sensitivity in detecting small physiological differences in the target metabolism. In comparison to the two-site exchange models, the multi site model yielded metabolic conversion rates with smaller bias and smaller standard deviation, as demonstrated in simulations with different signal-to-noise ratio. Pyruvate dose effects observed previously were confirmed and quantified through metabolic conversion rate values. Parameter interdependency allowed an accurate quantification and can therefore be useful for monitoring metabolic activity in different tissues

  1. Multisite Kinetic Modeling of 13C Metabolic MR Using [1-13C]Pyruvate

    Directory of Open Access Journals (Sweden)

    Pedro A. Gómez Damián

    2014-01-01

    Full Text Available Hyperpolarized 13C imaging allows real-time in vivo measurements of metabolite levels. Quantification of metabolite conversion between [1-13C]pyruvate and downstream metabolites [1-13C]alanine, [1-13C]lactate, and [13C]bicarbonate can be achieved through kinetic modeling. Since pyruvate interacts dynamically and simultaneously with its downstream metabolites, the purpose of this work is the determination of parameter values through a multisite, dynamic model involving possible biochemical pathways present in MR spectroscopy. Kinetic modeling parameters were determined by fitting the multisite model to time-domain dynamic metabolite data. The results for different pyruvate doses were compared with those of different two-site models to evaluate the hypothesis that for identical data the uncertainty of a model and the signal-to-noise ratio determine the sensitivity in detecting small physiological differences in the target metabolism. In comparison to the two-site exchange models, the multisite model yielded metabolic conversion rates with smaller bias and smaller standard deviation, as demonstrated in simulations with different signal-to-noise ratio. Pyruvate dose effects observed previously were confirmed and quantified through metabolic conversion rate values. Parameter interdependency allowed an accurate quantification and can therefore be useful for monitoring metabolic activity in different tissues.

  2. Simultaneous measurement of neuronal and glial metabolism in rat brain in vivo using co-infusion of [1,6- 13C 2]glucose and [1,2- 13C 2]acetate

    Science.gov (United States)

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

    2009-02-01

    In this work the feasibility of measuring neuronal-glial metabolism in rat brain in vivo using co-infusion of [1,6- 13C 2]glucose and [1,2- 13C 2]acetate was investigated. Time courses of 13C spectra were measured in vivo while infusing both 13C-labeled substrates simultaneously. Individual 13C isotopomers (singlets and multiplets observed in 13C spectra) were quantified automatically using LCModel. The distinct 13C spectral pattern observed in glutamate and glutamine directly reflected the fact that glucose was metabolized primarily in the neuronal compartment and acetate in the glial compartment. Time courses of concentration of singly and multiply-labeled isotopomers of glutamate and glutamine were obtained with a temporal resolution of 11 min. Although dynamic metabolic modeling of these 13C isotopomer data will require further work and is not reported here, we expect that these new data will allow more precise determination of metabolic rates as is currently possible when using either glucose or acetate as the sole 13C-labeled substrate.

  3. Estimation of glucose carbon recycling in children with glycogen storage disease: A 13C NMR study using [U-13C]glucose

    International Nuclear Information System (INIS)

    Kalderon, B.; Korman, S.H.; Gutman, A.; Lapidot, A.

    1989-01-01

    A stable isotope procedure to estimate hepatic glucose carbon recycling and thereby elucidate the mechanism by which glucose is produced in patients lacking glucose 6-phosphatase is described. A total of 10 studies was performed in children with glycogen storage disease type I (GSD-I) and type III (GSD-III) and control subjects. A primed dose-constant nasogastric infusion of D-[U- 13 C]glucose or an infusion diluted with nonlabeled glucose solution was administered following different periods of fasting. Hepatic glucose carbon recycling was estimated from 13 C NMR spectra. The values obtained for GSD-I patients coincided with the standard [U- 13 C]glucose dilution curve. These results indicate that the plasma glucose of GSD-I subjects comprises only a mixture of 99% 13 C-enriched D-[U- 13 C]glucose and unlabeled glucose but lacks any recycled glucose. Significantly different glucose carbon recycling values were obtained for two GSD-III patients in comparison to GSD-I patients. The results eliminate a mechanism for glucose production in GSD-I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity would result in endogenous glucose production of non- 13 C-labeled and nonrecycled glucose carbon, as was found in this study. In GSD-III patients gluconeogenesis is suggested as the major route for endogenous glucose synthesis. The contribution of the triose-phosphate pathway in these patients has been determined

  4. Evolution of E. coli on [U-13C] Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology

    DEFF Research Database (Denmark)

    Sandberg, Troy E.; Long, Christopher P.; Gonzalez, Jacqueline E.

    2016-01-01

    13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia...

  5. Synthesis of 1-13C-1-indanone and 2-13C-1,2,3,4-tetrahydroquinoline

    International Nuclear Information System (INIS)

    Pickering, R.E.; Wysocki, M.A.; Eisenbraun, E.J.

    1985-01-01

    The synthesis of 2- 13 C-1,2,3,4-tetrahydroquinoline (5) via 1- 13 C-3-phenylpropanoic acid (1), 1- 13 C-1-indanone (2), 1- 13 C-1-indanone hydrazone (3) and 2- 13 C-3,4-dihydro-2(1H)-quinolinone (4) proceeded in 78, 96, 95, 79, and 85% individual yields respectively for 1, 2, 3, 4, 5 and 61% overall yield of the latter from 1. (author)

  6. Neutron orbital radii in {sup 13} C; Radios orbitales neutronicos en {sup 13} C

    Energy Technology Data Exchange (ETDEWEB)

    Aguilera R, E.F.; Murillo, G.; Ramirez, J.J.; Avila, O.L. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    1988-01-15

    In this work its were carried out experimental measurements of the reaction {sup 12}C(d,p) {sup 13}C at low energy. Preliminary results of a DWBA analysis of the data are presented, and the possibility of using this reaction to obtain the orbital radius of the transferred neutron is investigated. (Author)

  7. Solid-state NMR detection of 14N-13C dipolar couplings between amino acid side groups provides constraints on amyloid fibril architecture.

    Science.gov (United States)

    Middleton, David A

    2011-02-01

    Solid-state nuclear magnetic resonance (SSNMR) is a powerful technique for the structural analysis of amyloid fibrils. With suitable isotope labelling patterns, SSNMR can provide constraints on the secondary structure, alignment and registration of β-strands within amyloid fibrils and identify the tertiary and quaternary contacts defining the packing of the β-sheet layers. Detection of (14)N-(13)C dipolar couplings may provide potentially useful additional structural constraints on β-sheet packing within amyloid fibrils but has not until now been exploited for this purpose. Here a frequency-selective, transfer of population in double resonance SSNMR experiment is used to detect a weak (14)N-(13)C dipolar coupling in amyloid-like fibrils of the peptide H(2)N-SNNFGAILSS-COOH, which was uniformly (13)C and (15)N labelled across the four C-terminal amino acids. The (14)N-(13)C interatomic distance between leucine and asparagine side groups is constrained between 2.4 and 3.8 Å, which allows current structural models of the β-spine arrangement within the fibrils to be refined. This procedure could be useful for the general structural analysis of other proteins in condensed phases and environments, such as biological membranes. Copyright © 2011 John Wiley & Sons, Ltd.

  8. Applications of stable isotopes of 2H, 13C and 15N to clinical problems

    International Nuclear Information System (INIS)

    Klein, P.D.; Szczepanik, P.A.; Hachey, D.L.

    1974-01-01

    The function of the Argonne Program is to provide synthetic, analytical instrumental capability in a core facility for the clinical investigator who needs to use 2 H, 13 C, or 15 N labelled compounds for metabolic or clinical research on pregnant women, newborn infants, young children, or for mass screening. To carry out such application development, there were six stages which were recurrent steps in every application. Five fundamental strategies should be adopted to establish the use of stable isotopes in clinical work. The instrument required for measurements was a combined gas chromatograph-mass spectrometer, and its use was schematically illustrated. Some of the successful experiences with compounds labelled by stable isotopes, such as deuterium labelled chenodeoxycholic acid, and respective 13 C and 15 N-labelled glycine were described. Deutrium labelled bile acid enabled easy and safe determination of the size of the bile acid pool and the replacement rate, providing clearer diagnoses for cholestatic liver disease and gallstones. 13 C and 15 N labelled compounds were used in clinical studies, of children with genetic disorders of amino acid metabolism, i.e., non ketotic hyperflycinemia, B 12 -responsive methyl malonic acidemia, and Lesch-Nyhan syndrome. 15 N-labelled glycine was also studied in a child with Lesch-Nyhan syndrome. (Mukohata, S.)

  9. Synthesis of [2-13C, 2-14C] 2-aminoethanol, [1-13C, 1-14C] 2-chloroethylamine, N,N'-bis([1-13C, 1-14C] 2-chloroethyl)-N-nitrosourea(BCNU) and N-([1-13C, 1-14C] 2-chloroethyl)-N-nitrosourea(CNU)

    International Nuclear Information System (INIS)

    Narayan, R.; Chang, C-j.

    1982-01-01

    [2- 13 C, 2- 14 C]2-Aminoethanol hydrochloride was prepared in good yield from Na*CN in a two step sequence by first converting the Na*CN to OHCH 2 *CN and then reducing the nitrile directly with a solution of borane-tetrahydrofuran complex. The reaction procedure was simple and the pure product could be obtained readily. Using this specifically labelled precursor, the synthesis of [1- 13 C, 1- 14 C]2-chloroethylamine hydrochloride, N-([1- 13 C, 1- 14 C]2-chloroethyl)-N-nitrosourea(CNU) and N,N'-bis([1- 13 C, 1- 14 C]2-chloroethyl)-N-nitrosourea(BCNU) in good yield without isotope scrambling was also reported. (author)

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

    Science.gov (United States)

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

    2015-10-01

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

  11. Structural characterization of supramolecular assemblies by {sup 13}C spin dilution and 3D solid-state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Habenstein, Birgit; Loquet, Antoine; Giller, Karin; Becker, Stefan; Lange, Adam, E-mail: adla@nmr.mpibpc.mpg.de [Max Planck Institute for Biophysical Chemistry, Department of NMR-based Structural Biology (Germany)

    2013-01-15

    {sup 13}C spin diluted protein samples can be produced using [1-{sup 13}C] and [2-{sup 13}C]-glucose (Glc) carbon sources in the bacterial growth medium. The {sup 13}C spin dilution results in favorable {sup 13}C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-{sup 13}C]- and [2-{sup 13}C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-{sup 13}C]-glycerol ({sup 13}C labeled C{alpha} sites on a {sup 12}C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17-26, 2011 ). Inspired by this approach and our own recent results using [2-{sup 13}C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of {sup 13}C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.

  12. Synthesis of (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)olivetolic acid, methyl (1'-/sup 13/C)olivetolate and (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)cannabigerolic acid

    Energy Technology Data Exchange (ETDEWEB)

    Porwoll, J P; Leete, E [Minnesota Univ., Minneapolis (USA). Dept. of Chemistry

    1985-03-01

    Potential advanced intermediates in the biosynthesis of delta/sup 9/-tetrahydrocannabinol, the major psychoactive principle of marijuana, have been synthesized labeled with two contiguous /sup 13/C atoms and /sup 14/C. Methyl (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)olivetolate was prepared from lithium (/sup 13/C/sub 2/)acetylide and dimethyl (2-/sup 14/C)malonate. Reaction with geranyl bromide afforded methyl (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)cannabigerolate, and hydrolysis of these methyl esters with lithium propyl mercaptide yielded the corresponding labeled acids. The /sup 13/C-/sup 13/C couplings observable in the /sup 13/C NMR spectra of these /sup 13/C-enriched compounds and their synthetic precursors are recorded. Methyl (1'-/sup 14/C)olivetolate was prepared from /sup 13/CO/sub 2/ to confirm assignments of the /sup 13/C chemical shifts in the pentyl side chain of these compounds.

  13. Synthesis of (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)olivetolic acid, methyl (1'-/sup 13/C)olivetolate and (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)cannabigerolic acid

    Energy Technology Data Exchange (ETDEWEB)

    Porwoll, J.P.; Leete, E. (Minnesota Univ., Minneapolis (USA). Dept. of Chemistry)

    1985-03-01

    Potential advanced intermediates in the biosynthesis of delta/sup 9/-tetrahydrocannabinol, the major psychoactive principle of marijuana, have been synthesized labeled with two contiguous /sup 13/C atoms and /sup 14/C. Methyl (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)olivetolate was prepared from lithium (/sup 13/C/sub 2/)acetylide and dimethyl (2-/sup 14/C)malonate. Reaction with geranyl bromide afforded methyl (5,6-/sup 13/C/sub 2/, 1-/sup 14/C)cannabigerolate, and hydrolysis of these methyl esters with lithium propyl mercaptide yielded the corresponding labeled acids. The /sup 13/C-/sup 13/C couplings observable in the /sup 13/C NMR spectra of these /sup 13/C-enriched compounds and their synthetic precursors are recorded. Methyl (1'-/sup 14/C)olivetolate was prepared from /sup 13/CO/sub 2/ to confirm assignments of the /sup 13/C chemical shifts in the pentyl side chain of these compounds.

  14. Tracing carbon flow from microphytobenthos to major bacterial groups in an intertidal marine sediment by using an in situ 13C pulse-chase method

    NARCIS (Netherlands)

    Miyatake, T.; Moerdijk-Poortvliet, T.C.W.; Stal, L.J.; Boschker, H.T.S.

    2014-01-01

    Carbon flow from benthic diatoms to heterotrophic bacterial was traced in an intertidal sediment for 5 consecutive days. 13C-labeled bicarbonate was sprayed onto the sediment surface during low tide and 13C-label incorporation in major carbon pools, intermediate metabolites, and biomarkers were

  15. Detection of the sulfhydryl groups in proteins with slow hydrogen exchange rates and determination of their proton/deuteron fractionation factors using the deuterium-induced effects on the 13C(beta) NMR signals.

    Science.gov (United States)

    Takeda, Mitsuhiro; Jee, JunGoo; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-05-05

    A method for identifying cysteine (Cys) residues with sulfhydryl (SH) groups exhibiting slow hydrogen exchange rates has been developed for proteins in aqueous media. The method utilizes the isotope shifts of the C(beta) chemical shifts induced by the deuteration of the SH groups. The 18.2 kDa E. coli peptidyl prolyl cis-trans isomerase b (EPPIb), which was selectively labeled with [3-(13)C;3,3-(2)H(2)]Cys, showed much narrower line widths for the (13)C(beta) NMR signals, as compared to those of the proteins labeled with either [3-(13)C]Cys or (3R)-[3-(13)C;3-(2)H]Cys. The (13)C(beta) signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-(13)C;3,3-(2)H(2)]Cys, split into four signals in H(2)O/D(2)O (1:1) at 40 degrees C and pH 7.5, indicating that the exchange rates of the side-chain SH's and the backbone amides are too slow to average the chemical shift differences of the (13)C(beta) signals, due to the two- and three-bond isotope shifts. By virtue of the well-separated signals, the proton/deuteron fractional factors for both the SH and amide groups of the two Cys residues in EPPIb could be directly determined, as approximately 0.4-0.5 for [SD]/[SH] and 0.9-1.0 for [ND]/[NH], by the relative intensities of the NMR signals for the isotopomers. The proton NOE's of the two slowly exchanging SH's were clearly identified in the NOESY spectra and were useful for the determining the local structure of EPPIb around the Cys residues.

  16. Detailed {sup 1}H and {sup 13}C NMR spectral data assignment for two dihydrobenzofuran neolignans

    Energy Technology Data Exchange (ETDEWEB)

    Medeiros, Talita C.T.; Dias, Herbert J.; Crotti, Antônio E.M., E-mail: millercrotti@ffclrp.usp.br [Universidade de São Paulo (USP), Ribeirão Preto, SP (Brazil). Faculdade de Filosofia, Ciências e Letras. Departamento de Química

    2016-07-01

    In this work we present a complete proton ({sup 1}H) and carbon 13 ({sup 13}C) nuclear magnetic resonance (NMR) spectral analysis of two synthetic dihydrofuran neolignans (±)-trans-dehydrodicoumarate dimethyl ester and (±)-trans-dehydrodiferulate dimethyl ester. Unequivocal assignments were achieved by 1 H NMR, proton decoupled {sup 13}C ({sup 13}C{"1H}) NMR spectra, gradient-selected correlation spectroscopy (gCOSY), J-resolved, gradient-selected heteronuclear multiple quantum coherence (gHMQC), gradient-selected heteronuclear multiple bond coherence (gHMBC) and nuclear Overhauser effect spectroscopy (NOESY) experiments. All hydrogen coupling constants were measured, clarifying all the hydrogen signals multiplicities. Computational methods were also used to simulate the {sup 1}H and {sup 13}C chemical shifts and showed good agreement with the trans configuration of the substituents at C{sub 7} and C{sub 8}. (author)

  17. Detailed 1H and 13C NMR spectral data assignment for two dihydrobenzofuran neolignans

    International Nuclear Information System (INIS)

    Medeiros, Talita C.T.; Dias, Herbert J.; Crotti, Antônio E.M.

    2016-01-01

    In this work we present a complete proton ( 1 H) and carbon 13 ( 13 C) nuclear magnetic resonance (NMR) spectral analysis of two synthetic dihydrofuran neolignans (±)-trans-dehydrodicoumarate dimethyl ester and (±)-trans-dehydrodiferulate dimethyl ester. Unequivocal assignments were achieved by 1 H NMR, proton decoupled 13 C ( 13 C{ 1 H}) NMR spectra, gradient-selected correlation spectroscopy (gCOSY), J-resolved, gradient-selected heteronuclear multiple quantum coherence (gHMQC), gradient-selected heteronuclear multiple bond coherence (gHMBC) and nuclear Overhauser effect spectroscopy (NOESY) experiments. All hydrogen coupling constants were measured, clarifying all the hydrogen signals multiplicities. Computational methods were also used to simulate the 1 H and 13 C chemical shifts and showed good agreement with the trans configuration of the substituents at C 7 and C 8 . (author)

  18. Search for biological effects of 13C-enrichment in developing mammalian systems

    International Nuclear Information System (INIS)

    Gregg, C.; Ott, D.; Deaven, L.; Spielmann, H.; Krowke, R.; Neubert, D.

    1975-01-01

    Increasing diagnostic use of stable isotopes, especially in children and pregnant women, enhances the importance of studies on the biological isotope effects in sensitive mammalian systems. Experimental data on animal systems are meager. The mouse embryos was studied at various stages and mouse limb buds were studied in organ culture. Limb bud development in vitro was unaffected by incubation with 82 mol percent 13 C-glucose as judged by either morphological or biochemical criteria. Of 271 preimplantation embryos incubated in vitro, 95.2 percent developed normally; in 13 C-enriched medium, 96.5 percent showed normal development. 13 C-Enrichment of the embryos in vitro is over 60 percent. Administration of 1.2 g glucose-U- 13 C to pregnant mice during organogenesis leads to enrichment of maternal liver glycogen to over 17 mol percent 13 C, about one-third this level in the embryo, and a lower level in maternal blood. The absolute 13 C content of the embryo continues to increase for several days after the end of isotope administration, while the enrichment in maternal tissues falls. The lipid fraction of the fetus is most highly labeled shortly after the end of isotope administration []These studies on developing mammalian systems have not yet revealed any alteration of normal development due to stable isotope enrichment. (auth)

  19. Millimetre-wave spectrum of anti-13C1 and 13C2 isotopologues of ethanol

    International Nuclear Information System (INIS)

    Bouchez, Aurelia; Walters, Adam; Müller, Holger S.P.; Ordu, Matthias; Lewen, Frank; Koerber, Monika; Bottinelli, Sandrine; Endres, Christian P.; Schlemmer, Stephan

    2012-01-01

    The rotational spectra of the two monosubstituted 13 C isotopologues of the anti conformer of ethanol have been measured between 80-800 GHz using three different spectrometers at the Cologne Laboratory Astrophysics group. The dataset was constrained for fitting with a standard Watson-S reduction Hamiltonian by rejecting transitions from high-lying states showing significant perturbation with the gauche states and by averaging some small methyl torsional splits. This treatment is compatible with the needs for a first astrophysical research for which an appropriate set of predictions is given.

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

    Directory of Open Access Journals (Sweden)

    Belshoff Alex C

    2011-05-01

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

  1. The ratio of acetate-to-glucose oxidation in astrocytes from a single 13C NMR spectrum of cerebral cortex.

    Science.gov (United States)

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

    2015-01-01

    The (13) C-labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of (13) C-enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α-ketoglutarate into glutamate in neurons, and incorporation of α-ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single (13) C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate-to-glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2-(13) C]acetate and [1,6-(13) C]glucose, and proton decoupled (13) C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that (13) C-labeled acetate and glucose contributed approximately equally to acetyl-CoA (0.96) in astrocytes. As this method relies on a single (13) C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions. Differences in (13) C labeling of brain glutamate and glutamine have been attributed to metabolic compartmentation. The acetate:glucose ratio, introduced for description of a (13) C NMR (nuclear magnetic resonance) spectrum, is an index of glucose and acetate oxidation in brain tissue. A simple mathematical method is presented to quantify the ratio of acetate-to-glucose oxidation in astrocytes from a single NMR spectrum. As kinetic analysis is not required, the method is readily applicable to analysis of tissue extracts. α-KG = alpha-ketoglutarate; CAC = citric acid cycle; GLN = glutamine; GLU = glutamate. © 2014 International Society for Neurochemistry.

  2. Energy and traffic light labelling have no impact on parent and child fast food selection.

    Science.gov (United States)

    Dodds, Pennie; Wolfenden, Luke; Chapman, Kathy; Wellard, Lyndal; Hughes, Clare; Wiggers, John

    2013-10-25

    Labelling of food from fast food restaurants at point-of-purchase has been suggested as one strategy to reduce population energy consumption and contribute to reductions in obesity prevalence. The aim of this study was to examine the effects of energy and single traffic light labelling systems on the energy content of child and adult intended food purchases. The study employed a randomised controlled trial design. English speaking parents of children aged between three and 12 years were recruited from an existing research cohort. Participants were mailed one of three hypothetical fast food menus. Menus differed in their labelling technique- either energy labels, single traffic light labels, or a no-label control. Participants then completed a telephone survey which assessed intended food purchases for both adult and child. The primary trial outcome was total energy of intended food purchase. A total of 329 participants completed the follow-up telephone interview. Eighty-two percent of the energy labelling group and 96% of the single traffic light labelling group reported noticing labelling information on their menu. There were no significant differences in total energy of intended purchases of parents, or intended purchases made by parents for children, between the menu labelling groups, or between menu labelling groups by socio-demographic subgroups. This study provided no evidence to suggest that energy labelling or single traffic light labelling alone were effective in reducing the energy of fast food items selected from hypothetical fast food menus for purchase. Additional complementary public health initiatives promoting the consumption of healthier foods identified by labelling, and which target other key drivers of menu item selection in this setting may be required. Copyright © 2013. Published by Elsevier Ltd.

  3. Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

    Science.gov (United States)

    Timm, Kerstin N; Hartl, Johannes; Keller, Markus A; Hu, De-En; Kettunen, Mikko I; Rodrigues, Tiago B; Ralser, Markus; Brindle, Kevin M

    2015-12-01

    A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells. © 2014 Wiley Periodicals, Inc.

  4. Combined Hyperpolarized 13C-pyruvate MRS and 18F-FDG PET (HyperPET) Estimates of Glycolysis in Canine Cancer Patients

    DEFF Research Database (Denmark)

    Hansen, Adam E.; Gutte, Henrik; Holst, Pernille

    2018-01-01

    13C Magnetic Resonance Spectroscopy (MRS) using hyperpolarized 13C-labeled pyruvate as a substrate offers a measure of pyruvate-lactate interconversion and is thereby a marker of the elevated aerobic glycolysis (Warburg effect) generally exhibited by cancer cells. Here, we aim to compare hyperpol......13C Magnetic Resonance Spectroscopy (MRS) using hyperpolarized 13C-labeled pyruvate as a substrate offers a measure of pyruvate-lactate interconversion and is thereby a marker of the elevated aerobic glycolysis (Warburg effect) generally exhibited by cancer cells. Here, we aim to compare...

  5. Antiferromagnetic Ordering in Quasi-Triangular Localized Spin System, β'-Et2Me2P[Pd(dmit)2]2, Studied by 13C NMR

    Science.gov (United States)

    Otsuka, Kei; Iikubo, Hideaki; Kogure, Takayuki; Takano, Yoshiki; Hiraki, Ko-ichi; Takahashi, Toshihiro; Cui, Hengbo; Kato, Reizo

    2014-05-01

    We performed 13C NMR measurements of a selectively 13C isotope-labeled single-crystal sample of a frustrated spin system, β'-Et2Me2P[Pd(dmit)2]2. A long-range antiferromagnetic (AF) ordering below 17 K was confirmed by the observation of NMR spectrum broadening and well split resonance lines at lower temperatures. NMR spectra in the AF state can be well explained by a two sublattice model. From the analysis of the angular dependence of the NMR spectrum, we clarified the magnetic structure in the AF state, where the easy and hard axes are the crystallographic c*- and b-axes, respectively, and the effective localized moments are quite small, ˜0.28 μB/dimer. This suggests a strong quantum fluctuation effect due to magnetic frustrations in a quasi-triangular spin-1/2 system.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ahlner, Alexandra; Andresen, Cecilia; Khan, Shahid N. [Linköping University, Division of Chemistry, Department of Physics, Chemistry and Biology (Sweden); Kay, Lewis E. [The University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry, One King’s College Circle (Canada); Lundström, Patrik, E-mail: patlu@ifm.liu.se [Linköping University, Division of Chemistry, Department of Physics, Chemistry and Biology (Sweden)

    2015-07-15

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

  7. Label fusion based brain MR image segmentation via a latent selective model

    Science.gov (United States)

    Liu, Gang; Guo, Xiantang; Zhu, Kai; Liao, Hengxu

    2018-04-01

    Multi-atlas segmentation is an effective approach and increasingly popular for automatically labeling objects of interest in medical images. Recently, segmentation methods based on generative models and patch-based techniques have become the two principal branches of label fusion. However, these generative models and patch-based techniques are only loosely related, and the requirement for higher accuracy, faster segmentation, and robustness is always a great challenge. In this paper, we propose novel algorithm that combines the two branches using global weighted fusion strategy based on a patch latent selective model to perform segmentation of specific anatomical structures for human brain magnetic resonance (MR) images. In establishing this probabilistic model of label fusion between the target patch and patch dictionary, we explored the Kronecker delta function in the label prior, which is more suitable than other models, and designed a latent selective model as a membership prior to determine from which training patch the intensity and label of the target patch are generated at each spatial location. Because the image background is an equally important factor for segmentation, it is analyzed in label fusion procedure and we regard it as an isolated label to keep the same privilege between the background and the regions of interest. During label fusion with the global weighted fusion scheme, we use Bayesian inference and expectation maximization algorithm to estimate the labels of the target scan to produce the segmentation map. Experimental results indicate that the proposed algorithm is more accurate and robust than the other segmentation methods.

  8. Selective detection of labeled DNA using an air-clad photonic crystal fiber

    DEFF Research Database (Denmark)

    Jensen, Jesper Bo Damm; Hoiby, P.E.; Pedersen, L.H.

    2004-01-01

    Demonstration of selective detection of fluorophore labeled DNA by hybridization inside the air holes of a photonic crystal fiber A laser exposes the fiber from the side and the emitted fluorescence tunnels into the core.......Demonstration of selective detection of fluorophore labeled DNA by hybridization inside the air holes of a photonic crystal fiber A laser exposes the fiber from the side and the emitted fluorescence tunnels into the core....

  9. [2,4-(13)C]β-hydroxybutyrate metabolism in astrocytes and C6 glioblastoma cells.

    Science.gov (United States)

    Eloqayli, Haytham; Melø, Torun M; Haukvik, Anne; Sonnewald, Ursula

    2011-08-01

    This study was undertaken to determine if the ketogenic diet could be useful for glioblastoma patients. The hypothesis tested was whether glioblastoma cells can metabolize ketone bodies. Cerebellar astrocytes and C6 glioblastoma cells were incubated in glutamine and serum free medium containing [2,4-(13)C]β-hydroxybutyrate (BHB) with and without glucose. Furthermore, C6 cells were incubated with [1-(13)C]glucose in the presence and absence of BHB. Cell extracts were analyzed by mass spectrometry and media by (1)H magnetic resonance spectroscopy and HPLC. Using [2,4-(13)C]BHB and [1-(13)C]glucose it could be shown that C6 cells, in analogy to astrocytes, had efficient mitochondrial activity, evidenced by (13)C labeling of glutamate, glutamine and aspartate. However, in the presence of glucose, astrocytes were able to produce and release glutamine, whereas this was not accomplished by the C6 cells, suggesting lack of anaplerosis in the latter. We hypothesize that glioblastoma cells kill neurons by not supplying the necessary glutamine, and by releasing glutamate.

  10. Polymeric proanthocyanidins 13C NMR studies of procyanidins

    Science.gov (United States)

    Lawrence J. Porter; Roger H. Newman; Lai Yeap Foo; Herbert Wong; Richard W. Hemingway

    1982-01-01

    Proanthocyanidin polymers have been shown to consist entirely of flavan-3-ol units by a combination of techniques including 13C n.m.r. spectroscopy. The 13C n.m.r. spectra of the polymers and related molecules are now considered in more detail. Prior to this study UC n.m.r. data has been published of procyanidins and...

  11. 13C-NMR assignment, structure, and dynamics of deoxyoligonucleotides

    International Nuclear Information System (INIS)

    Zanatta, N.; Borer, P.N.; Levy, G.C.

    1986-01-01

    The unique spectral properties of 13 C-NMR for studying nucleic acids and some of the important features of 13 C-NMR in oligonucleotide studies are demostrated. The main difficulty in studying oligonucleotides by 13 C-NMR and recent improvements in NMR instrumentation and advances in oligonucleotide synthesis are presented. The high resolution 13 C-NMR spectra, T 1 relaxation times and NOEs were measured for duplex of the self-complementary oligo-DNAs: d(CG) 3 and d(GGTATACC) are studied. The target of this study is to developed a systematic 13 C-NMR spectral assignment and to investigate the structure and dynamics of these two sequences by this techniques. (M.J.C.) [pt

  12. Characterization of methacetin-methoxy-"1"3C

    International Nuclear Information System (INIS)

    Lu Weijing; Lu Hao; Yang Weicheng; Liu Weixia; Li Shuai; Xu Zhongjie; Guan Liang; Zhu Chengmo; Chen Suyun; Jiang Lei

    2010-01-01

    Methacetin-methoxy-"1"3C was synthesized by using methanol-"1"3C with a novel method, and the characterization of it was performed using HPLC, LC-MS and "1HMNR. The results indicated that the synthetic was right. And the yield of methacetin-methoxy-"1"3C was 70.0% with 99% "1"3C abundance and 99.8% purity. Compared with the classical method, there was more benefit. The methacetin "1"3C-breath test was performed with the synthetic on the live mice, which showed a precise reflection of alteration of liver function in liver injury and functional recovery. (authors)

  13. NMR study of the 1-13C glucose colon bacterial metabolism

    International Nuclear Information System (INIS)

    Briet, F.; Flourie, B.; Pochart, P.; Rambaud, J.C.; Desjeux, J.F.; Dallery, L.; Grivet, J.P.

    1994-01-01

    The aim of the study is to examine in-vitro and by nuclear magnetic resonance the biological pathways for the fermentation of the 1- 13 C labelled glucose (99 atoms percent) by human colon bacteria. The preparation of the bacterial suspension and the glucose degradation kinetics are presented; the NMR analysis sensitivity and quantification features are discussed and results are presented. 2 figs., 1 ref

  14. A perspective on tritium versus carbon-14: ensuring optimal label selection in pharmaceutical research and development.

    Science.gov (United States)

    Krauser, Joel A

    2013-01-01

    Tritium ((3) H) and carbon-14 ((14) C) labels applied in pharmaceutical research and development each offer their own distinctive advantages and disadvantages coupled with benefits and risks. The advantages of (3) H have a higher specific activity, shorter half-life that allows more manageable waste remediation, lower material costs, and often more direct synthetic routes. The advantages of (14) C offer certain analytical benefits and less potential for label loss. Although (3) H labels offer several advantages, they might be overlooked as a viable option because of the concerns about its drawbacks. A main drawback often challenged is metabolic liability. These drawbacks, in some cases, might be overstated leading to underutilization of a perfectly viable option. As a consequence, label selection may automatically default to (14) C, which is a more conservative approach. To challenge this '(14) C-by-default' approach, pharmaceutical agents with strategically selected (3) H-labeling positions based on non-labeled metabolism data have been successfully implemented and evaluated for (3) H loss. From in-house results, the long term success of projects clearly would benefit from a thorough, objective, and balanced assessment regarding label selection ((3) H or (14) C). This assessment should be based on available project information and scientific knowledge. Important considerations are project applicability (preclinical and clinical phases), synthetic feasibility, costs, and timelines. Copyright © 2013 John Wiley & Sons, Ltd.

  15. Characterization of water in hydrated Bombyx mori silk fibroin fiber and films by 2H NMR relaxation and 13C solid state NMR.

    Science.gov (United States)

    Asakura, Tetsuo; Isobe, Kotaro; Kametani, Shunsuke; Ukpebor, Obehi T; Silverstein, Moshe C; Boutis, Gregory S

    2017-03-01

    The mechanical properties of Bombyx mori silk fibroin (SF), such as elasticity and tensile strength, change remarkably upon hydration. However, the microscopic interaction with water is not currently well understood on a molecular level. In this work, the dynamics of water molecules interacting with SF was studied by 2 H solution NMR relaxation and exchange measurements. Additionally, the conformations of hydrated [3- 13 C]Ala-, [3- 13 C]Ser-, and [3- 13 C]Tyr-SF fibers and films were investigated by 13 C DD/MAS NMR. Using an inverse Laplace transform algorithm, we were able to identify four distinct components in the relaxation times for water in SF fiber. Namely, A: bulk water outside the fiber, B: water molecules trapped weakly on the surface of the fiber, C: bound water molecules located in the inner surface of the fiber, and D: bound water molecules located in the inner part of the fiber were distinguishable. In addition, four components were also observed for water in the SF film immersed in methanol for 30s, while only two components for the film immersed in methanol for 24h. The effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and non-crystalline domains of 13 C selectively labeled SF, respectively, could be determined independently. Our measurements provide new insight relating the characteristics of water and the hydration structure of silk, which are relevant in light of current interest in the design of novel silk-based biomaterials. The mechanical properties of Bombyx mori silk fibroin (SF) change remarkably upon hydration. However, the microscopic interaction between SF and water is not currently well understood on a molecular level. We were able to identify four distinct components in the relaxation times for water in SF fiber by 2 H solution NMR relaxation and exchange measurements. In addition, the effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and

  16. An improved synthesis of α-13C glycine and heteronuclear NMR studies of its incorporation into thioredoxin

    International Nuclear Information System (INIS)

    Wishart, D.S.; Sykes, B.D.; Richards, F.M.

    1992-01-01

    We present an improved method to easily prepare gram quantities of α- 13 C glycine beginning from K 13 CN. The four step synthesis involves the production of an N, N-diphenyl-cyanoformamidine intermediate through the coupling of cyanide to N, N-diphenylcarbodiimide. Subsequent reduction by LiAlH 4 and hydrolysis of the resulting amidine produces fully enriched α- 13 C labelled glycine with a 45-50% yield. This relatively fast and simple synthesis uses only commonly available compounds and requires no special equipment, making the process easy to perform in any well equipped biochemistry laboratory. We further demonstrate that the product may be used, without extensive purification, to specifically label bacterially expressed proteins (E. coli thioredoxin) through standard biosynthetic procedures. We also show that the 13 C glycine-labelled protein may be readily analyzed using commonly available heteronuclear NMR techniques. Complete assignments for all 9 glycines of native E. coli thoredoxin are presented. (Author)

  17. Whole-core analysis by 13C NMR

    International Nuclear Information System (INIS)

    Vinegar, H.J.; Tutunjian, P.N.; Edelstein, W.A.; Roemer, P.B.

    1991-01-01

    This paper reports on a whole-core nuclear magnetic resonance (NMR) system that was used to obtain natural abundance 13 C spectra. The system enables rapid, nondestructive measurements of bulk volume of movable oil, aliphatic/aromatic ratio, oil viscosity, and organic vs. carbonate carbon. 13 C NMR can be used in cores where the 1 H NMR spectrum is too broad to resolve oil and water resonances separately. A 5 1/4-in. 13 C/ 1 H NMR coil was installed on a General Electric (GE) CSI-2T NMR imager/spectrometer. With a 4-in.-OD whole core, good 13 C signal/noise ratio (SNR) is obtained within minutes, while 1 H spectra are obtained in seconds. NMR measurements have been made of the 13 C and 1 H density of crude oils with a wide range of API gravities. For light- and medium-gravity oils, the 13 C and 1 H signal per unit volume is constant within about 3.5%. For heavy crudes, the 13 C and 1 H density measured by NMR is reduced by the shortening of spin-spin relaxation time. 13 C and 1 H NMR spin-lattice relaxation times were measured on a suite of Cannon viscosity standards, crude oils (4 to 60 degrees API), and alkanes (C 5 through C 16 ) with viscosities at 77 degrees F ranging from 0.5 cp to 2.5 x 10 7 cp. The 13 C and 1 H relaxation times show a similar correlation with viscosity from which oil viscosity can be estimated accurately for viscosities up to 100 cp. The 13 C surface relaxation rate for oils on water-wet rocks is very low. Nonproton decoupled 13 C NMR is shown to be insensitive to kerogen; thus, 13 C NMR measures only the movable hydrocarbon content of the cores. In carbonates, the 13 C spectrum also contains a carbonate powder pattern useful in quantifying inorganic carbon and distinguishing organic from carbonate carbon

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  19. The use of isotope ratios (13C/12C) for vegetable oils authentication

    Science.gov (United States)

    Cristea, G.; Magdas, D. A.; Mirel, V.

    2012-02-01

    Stable isotopes are now increasingly used for the control of the geographical origin or authenticity of food products. The falsification may be more or less sophisticated and its sophistication as well as its costs increases with the improvement of analytical methods. In this study 22 vegetable oils (olive, sunflower, palm, maize) commercialized on Romanian market were investigated by mean of δ13C in bulk oil and the obtained results were compared with those reported in literature in order to check the labeling of these natural products. The obtained results were in the range of the mean values found in the literature for these types of oils, thus providing their accurate labeling.

  20. Metabolic origin of the {delta}{sup 13}C of respired CO{sub 2} in roots of Phaseolus vulgaris

    Energy Technology Data Exchange (ETDEWEB)

    Bathellier, C.; Tcherkez, G.; Cornic, G.; Ghashghaie, J. [Laboratoire d' Ecologie, Systematique et Evolution - ESE, CNRS-UMR 8079 - IFR 87, Batiment 362, Universite Paris-Sud, 91405-Orsay Cedex (France); Tcherkez, G. [Plateforme Metabolisme-Metabolome, IFR87 La Plante et son Environnement, Institut de Biotechnologie des Plantes, Batiment 630, Universite Paris-Sud, 91405-Orsay Cedex (France); Bligny, R.; Gout, E. [Laboratoire de Physiologie Cellulaire Vegetale CEA-Grenoble 17, rue des Martyrs, 38054 Grenoble Cedex 9 (France)

    2009-07-01

    - Root respiration is a major contributor to soil CO{sub 2} efflux, and thus an important component of ecosystem respiration. But its metabolic origin, in relation to the carbon isotope composition ({delta}{sup 13}C), remains poorly understood. - Here, {sup 13}C analysis was conducted on CO{sub 2} and metabolites under typical conditions or under continuous darkness in French bean (Phaseolus vulgaris) roots. {sup 13}C contents were measured either under natural abundance or following pulse-chase labeling with {sup 13}C-enriched glucose or pyruvate, using isotope ratio mass spectrometer (IRMS) and nuclear magnetic resonance (NMR) techniques. - In contrast to leaves, no relationship was found between the respiratory quotient and the {delta}{sup 13}C of respired CO{sub 2}, which stayed constant at a low value (c. -27.5 per thousand) under continuous darkness. With labeling experiments, it is shown that such a pattern is explained by the {sup 13}C-depleting effect of the pentose phosphate pathway; and the involvement of the Krebs cycle fueled by either the glycolytic input or the lipid/protein recycling. The anaplerotic phosphoenolpyruvate carboxylase (PEPc) activity sustained glutamic acid (Glu) synthesis, with no net effect on respired CO{sub 2}. - These results indicate that the root {delta}{sup 13}C signal does not depend on the availability of root respiratory substrates and it is thus plausible that, unless the {sup 13}C photosynthetic fractionation varies at the leaf level, the root {delta}{sup 13}C signal hardly changes under a range of natural environmental conditions. (authors)

  1. The metabolism of [3-(13)C]lactate in the rat brain is specific of a pyruvate carboxylase-deprived compartment.

    Science.gov (United States)

    Bouzier, A K; Thiaudiere, E; Biran, M; Rouland, R; Canioni, P; Merle, M

    2000-08-01

    Lactate metabolism in the adult rat brain was investigated in relation with the concept of lactate trafficking between astrocytes and neurons. Wistar rats were infused intravenously with a solution containing either [3-(13)C]lactate (534 mM) or both glucose (750 mM) and [3-(13)C]lactate (534 mM). The time courses of both the concentration and (13)C enrichment of blood glucose and lactate were determined. The data indicated the occurrence of [3-(13)C]lactate recycling through liver gluconeogenesis. The yield of glucose labeling was, however, reduced when using the glucose-containing infusate. After a 20-min or 1-h infusion, perchloric acid extracts of the brain tissue were prepared and subsequently analyzed by (13)C- and (1)H-observed/(13)C-edited NMR spectroscopy. The (13)C labeling of amino acids indicated that [3-(13)C]lactate was metabolized in the brain. Based on the alanine C3 enrichment, lactate contribution to brain metabolism amounted to 35% under the most favorable conditions used. By contrast with what happens with [1-(13)C]glucose metabolism, no difference in glutamine C2 and C3 labeling was evidenced, indicating that lactate was metabolized in a compartment deprived of pyruvate carboxylase activity. This result confirms, for the first time from an in vivo study, that lactate is more specifically a neuronal substrate.

  2. SORIOS – A method for evaluating and selecting environmental certificates and labels

    DEFF Research Database (Denmark)

    Kikkenborg Pedersen, Dennis; Dukovska-Popovska, Iskra; Ola Strandhagen, Jan

    2012-01-01

    This paper presents a general method for evaluating and selecting environmental certificates and labels for companies to use on products and services. The method is developed based on a case study using a Grounded Theory approach. The result is a generalized six-step method that features an initial...... searching strategy and an evaluation model that weighs the prerequisites, rewards and the organization of certificate or label against the strategic needs of a company....

  3. Iodine-labelling of albumin and fibrinogen and application in selecting implantable material-titanium oxide

    International Nuclear Information System (INIS)

    Liu Fangyan; Zhou Meiying; Zhang Feng

    1998-01-01

    Human serum albumin and fibrinogen were successfully labelled with 125 I. The labelled proteins were further applied to carry out a background study on the selection of the blood-compatible materials. The protein adsorption of four kinds of titanium oxide film was determined and compared. It was found that Sample B can adsorb more albumin and less fibrinogen than other three samples and hold the adsorbed albumin most stably

  4. Synthesis of labeled compounds

    International Nuclear Information System (INIS)

    Whaley, T.W.

    1977-01-01

    Intermediate compounds labeled with 13 C included methane, sodium cyanide, methanol, ethanol, and acetonitrile. A new method for synthesizing 15 N-labeled 4-ethylsulfonyl-1-naphthalene-sulfonamide was developed. Studies were conducted on pathways to oleic-1- 13 C acid and a second pathway investigated was based on carbonation of 8-heptadecynylmagnesium bromide with CO 2 to prepare sterolic acid. Biosynthetic preparations included glucose- 13 C from starch isolated from tobacco leaves following photosynthetic incubation with 13 CO 2 and galactose- 13 C from galactosylglycerol- 13 C from kelp. Research on growth of organisms emphasized photosynthetic growth of algae in which all cellular carbon is labeled. Preliminary experiments were performed to optimize the growth of Escherichia coli on sodium acetate- 13 C

  5. Biosynthesis of 24-methylsterols from (1,2-/sup 13/C/sub 2/) acetate; dihydrobrassicasterol and campesterol in tissue cultures of Physalis peruviana and ergosterol in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Seo, S.; Uomori, A.; Yoshimura, Y.; Takeda, K. (Shionogi and Co. Ltd., Osaka (Japan). Research Lab.)

    1984-09-01

    The /sup 13/C labelling patterns of the two methyl groups at C-25 of dihydrobrassicasterol biosynthesized from (1,2-/sup 13/C/sub 2/) acetate differ from those of campesterol and 24-methylenecholesterol obtained from cultured cells of Physalis peruviana and ergosterol from yeast.

  6. Biosynthesis of 24-methylsterols from [1,2-13C2] acetate; dihydrobrassicasterol and campesterol in tissue cultures of Physalis peruviana and ergosterol in yeast

    International Nuclear Information System (INIS)

    Seo, S.; Uomori, A.; Yoshimura, Y.; Takeda, K.

    1984-01-01

    The 13 C labelling patterns of the two methyl groups at C-25 of dihydrobrassicasterol biosynthesized from [1,2- 13 C 2 ] acetate differ from those of campesterol and 24-methylenecholesterol obtained from cultured cells of Physalis peruviana and ergosterol from yeast. (author)

  7. 13C-NMR of diterpenes with pimarane skeleton

    International Nuclear Information System (INIS)

    Garcez, W.S.; Pereira, A.L.; Silva Queiroz, P.P. da; Silva, R.S. da; Valente, L.M.M.; Peixoto, E.M.; Cunha Pinto, A. da

    1981-01-01

    The effect of substituent groups on the chemical shift of carbons using nuclear magnetic resonance spectra of carbon 13 ( 13 C-NMR) is discussed. Diterpenes having pimarane skeleton, isolated from plants of Velloziaceae family are analysed. (ARHC) [pt

  8. Structural properties of carbon nanotubes derived from 13C NMR

    KAUST Repository

    Abou-Hamad, E.; Babaa, M.-R.; Bouhrara, M.; Kim, Y.; Saih, Y.; Dennler, S.; Mauri, F.; Basset, Jean-Marie; Goze-Bac, C.; Wå gberg, T.

    2011-01-01

    We present a detailed experimental and theoretical study on how structural properties of carbon nanotubes can be derived from 13C NMR investigations. Magic angle spinning solid state NMR experiments have been performed on single- and multiwalled

  9. Revisiting the metathesis of 13C-monolabeled ethane

    KAUST Repository

    Maury, Olivier; Lefort, Laurent; Vidal, Vé ronique; Thivolle-Cazat, Jean; Basset, Jean-Marie

    2010-01-01

    The metathesis of 13C-monolabeled ethane leads to the parallel occurrence of degenerate and productive reactions, affording the statistical distribution of the various product isotopomers, which can be rationalized in terms of a mechanistic reaction

  10. Plasma incorporation, apparent retroconversion and β-oxidation of 13C-docosahexaenoic acid in the elderly

    Directory of Open Access Journals (Sweden)

    Brenna J Thomas

    2011-01-01

    Full Text Available Abstract Background Higher fish or higher docosahexaenoic acid (DHA intake normally correlates positively with higher plasma DHA level, but recent evidence suggests that the positive relationship between intake and plasma levels of DHA is less clear in the elderly. Methods We compared the metabolism of 13C-DHA in six healthy elderly (mean - 77 y old and six young adults (mean - 27 y old. All participants were given a single oral dose of 50 mg of uniformly labelled 13C-DHA. Tracer incorporation into fatty acids of plasma triglycerides, free fatty acids, cholesteryl esters and phospholipids, as well as apparent retroconversion and β-oxidation of 13C-DHA were evaluated 4 h, 24 h, 7d and 28d later. Results Plasma incorporation and β-oxidation of 13C-DHA reached a maximum within 4 h in both groups, but 13C-DHA was transiently higher in all plasma lipids of the elderly 4 h to 28d later. At 4 h post-dose, 13C-DHA β-oxidation was 1.9 times higher in the elderly, but over 7d, cumulative β-oxidation of 13C-DHA was not different in the two groups (35% in the elderly and 38% in the young. Apparent retroconversion of 13C-DHA was well below 10% of 13C-DHA recovered in plasma at all time points, and was 2.1 times higher in the elderly 24 h and 7d after tracer intake. Conclusions We conclude that 13C-DHA metabolism changes significantly during healthy aging. Since DHA is a potentially important molecule in neuro-protection, these changes may be relevant to the higher vulnerability of the elderly to cognitive decline.

  11. Volumetric spiral chemical shift imaging of hyperpolarized [2-(13) c]pyruvate in a rat c6 glioma model.

    Science.gov (United States)

    Park, Jae Mo; Josan, Sonal; Jang, Taichang; Merchant, Milton; Watkins, Ron; Hurd, Ralph E; Recht, Lawrence D; Mayer, Dirk; Spielman, Daniel M

    2016-03-01

    MRS of hyperpolarized [2-(13)C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13)C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13)C]pyruvate in glioma-bearing brain. Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13)C]pyruvate, [2-(13)C]lactate, and [5-(13)C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13)C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13)C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13)C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. Robust volumetric imaging with hyperpolarized [2-(13)C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate. © 2015 Wiley Periodicals, Inc.

  12. Identifying inter-residue resonances in crowded 2D {sup 13}C-{sup 13}C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Miao Yimin; Cross, Timothy A. [Florida State University, Department of Chemistry and Biochemistry (United States); Fu Riqiang, E-mail: rfu@magnet.fsu.edu [National High Magnet Field Lab (United States)

    2013-07-15

    The feasibility of using difference spectroscopy, i.e. subtraction of two correlation spectra at different mixing times, for substantially enhanced resolution in crowded two-dimensional {sup 13}C-{sup 13}C chemical shift correlation spectra is presented. With the analyses of {sup 13}C-{sup 13}C spin diffusion in simple spin systems, difference spectroscopy is proposed to partially separate the spin diffusion resonances of relatively short intra-residue distances from the longer inter-residue distances, leading to a better identification of the inter-residue resonances. Here solid-state magic-angle-spinning NMR spectra of the full length M2 protein embedded in synthetic lipid bilayers have been used to illustrate the resolution enhancement in the difference spectra. The integral membrane M2 protein of Influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Based on known amino acid resonance assignments from amino acid specific labeled samples of truncated M2 sequences or from time-consuming 3D experiments of uniformly labeled samples, some inter-residue resonances of the full length M2 protein can be identified in the difference spectra of uniformly {sup 13}C labeled protein that are consistent with the high resolution structure of the M2 (22-62) protein (Sharma et al., Science 330(6003):509-512, 2010)

  13. Migration and deposition of 13C in the full-tungsten ASDEX Upgrade tokamak

    International Nuclear Information System (INIS)

    Hakola, A; Aho-Mantila, L; Groth, M; Kurki-Suonio, T; Makkonen, T; Likonen, J; Koivuranta, S; Krieger, K; Mayer, M; Mueller, H W; Neu, R; Rohde, V

    2010-01-01

    The migration of carbon in low-density, low-confinement plasmas of ASDEX Upgrade was studied by injecting 13 C into the main chamber of the torus at the end of the 2007 experimental campaign. A selection of standard tungsten-coated lower-divertor and main-chamber tiles as well as a complete set of lower-divertor tiles with an uncoated poloidal marker stripe were removed from one poloidal cross section and analysed using secondary ion mass spectrometry. The poloidal deposition profiles of 13 C on both the tungsten-coated tiles and on the uncoated graphite areas of the marker tiles were measured and compared. For the W-coated lower-divertor tiles, 13 C was deposited mainly on the high-field side tiles, while barely detectable amounts of 13 C were observed on low-field side samples. In contrast, on the uncoated marker stripes the deposition was equally pronounced in the high-field and low-field side divertor. The marker-tile results are in agreement with those obtained from graphite tiles after the 2003 and 2005 13 C experiments in ASDEX Upgrade. In the case of W-coated tiles, the 13 C measurements were complemented by determining the total amount of deposited carbon ( 12 C) on the tiles, which also shows strong deposition at the inner parts of the lower divertor. The estimated deposition of 13 C on W at the divertor areas was less than 1.5% of the injected amount of 13 C atoms. The 13 C analyses of the main-chamber tiles and small silicon samples mounted in remote areas revealed significant deposition in the upper divertor, in upper parts of the heat shield, in the limiter region close to the injection valve, and below the roof baffle. Approximately 8% of the injected 13 C is estimated to have accumulated in these regions. Possible reasons for the different deposition patterns on W and on graphite in different regions of the torus are discussed.

  14. 13C NMR and relaxation studies of the nanomagnet Mn12-acetate

    Science.gov (United States)

    Achey, Randall M.; Kuhns, Philip L.; Reyes, Arneil P.; Moulton, William G.; Dalal, Naresh S.

    2001-08-01

    The nanomagnet [Mn12O12(CH3COO)16(H2O)4].2CH3COOH.4H2O, also known as Mn12, has been synthesized with 13C labeling at the CH3 groups, and investigated by 13C NMR at fields up to 23 T. Using oriented samples, it is possible to resolve four distinct 13C peaks at room temperature, located on both sides of the unshifted Larmor frequency. These peaks were assigned to the four hyperfine-shifted, magnetically inequivalent sets of 13CH3 groups in the Mn12 lattice, based on a comparison with the crystal structure and point-dipole and spin-density calculations. These results establish that the unpaired electron spin density of the S=10 system in this cluster extends over the entire molecular framework, not just the core. These results are discussed in relationship to inelastic neutron scattering measurements. The temperature and field dependence of the 13C nuclear-spin-lattice-relaxation time T1 on the least shifted peak was measured. A single weakly field-dependent minimum at about 60 K is observed in the temperature dependence of the measured T1. The relaxation mechanism responsible for the T1 minimum is ascribed mainly to hindered rotation of the methyl group of the acetate ligand at higher temperature, and to electronic spin fluctuations at lower temperature.

  15. Minimally invasive (13)C-breath test to examine phenylalanine metabolism in children with phenylketonuria.

    Science.gov (United States)

    Turki, Abrar; Murthy, Gayathri; Ueda, Keiko; Cheng, Barbara; Giezen, Alette; Stockler-Ipsiroglu, Sylvia; Elango, Rajavel

    2015-01-01

    Phenylketonuria (PKU) is an autosomal recessive disorder caused by deficiency of hepatic phenylalanine hydroxylase (PAH) leading to increased levels of phenylalanine in the plasma. Phenylalanine levels and phenylalanine hydroxylase (PAH) activity monitoring are currently limited to conventional blood dot testing. 1-(13)C-phenylalanine, a stable isotope can be used to examine phenylalanine metabolism, as the conversion of phenylalanine to tyrosine occurs in vivo via PAH and subsequently releases the carboxyl labeled (13)C as (13)CO2 in breath. Our objective was to examine phenylalanine metabolism in children with PKU using a minimally-invasive 1-(13)C-phenylalanine breath test ((13)C-PBT). Nine children (7 M: 2 F, mean age 12.5 ± 2.87 y) with PKU participated in the study twice: once before and once after sapropterin supplementation. Children were provided 6 mg/kg oral dose of 1-(13)C-phenylalanine and breath samples were collected at 20 min intervals for a period of 2h. Rate of CO2 production was measured at 60 min post-oral dose using indirect calorimetry. The percentage of 1-(13)C-phenylalanine exhaled as (13)CO2 was measured over a 2h period. Prior to studying children with PKU, we tested the study protocol in healthy children (n = 6; 4M: 2F, mean age 10.2 ± 2.48 y) as proof of principle. Production of a peak enrichment (Cmax) of (13)CO2 (% of dose) in all healthy children occurred at 20 min ranging from 17-29% of dose, with a subsequent return to ~5% by the end of 2h. Production of (13)CO2 from 1-(13)C-phenylalanine in all children with PKU prior to sapropterin treatment remained low. Following sapropterin supplementation for a week, production of (13)CO2 significantly increased in five children with a subsequent decline in blood phenylalanine levels, suggesting improved PAH activity. Sapropterin treatment was not effective in three children whose (13)CO2 production remained unchanged, and did not show a reduction in blood phenylalanine levels and improvement

  16. Fourier spectroscopy of the 12C2, 13C2, and 12C13C (0-0) swan bands

    International Nuclear Information System (INIS)

    Amiot, C.

    1983-01-01

    The (0-0) band of the C 2 Swan electronic system d 3 Pi/sub g/→a 3 Pi/sub u/ has been recorded by Fourier spectroscopy. The three isotopes species 12 C 2 , 13 C 2 , and 12 C 13 C were investigated. The observed wavenumbers were reduced to molecular parameters using a nonlinear least-square fitting procedure. Well-known perturbations at N' = 47 and N' = 51 again observed in the e 12 C 2 d 3 Pi/sub g/ (v = 0) level. Perturbations of the same kind are present in the 13 C 2 spectrum at N' = 34 and N' = 44,48,52. The 12 C 13 C spectrum exhibits in the observed spectral range a unique perturbation for N' = 41

  17. Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

    Science.gov (United States)

    Pham, Grace H; Ou, Weijia; Bursulaya, Badry; DiDonato, Michael; Herath, Ananda; Jin, Yunho; Hao, Xueshi; Loren, Jon; Spraggon, Glen; Brock, Ansgar; Uno, Tetsuo; Geierstanger, Bernhard H; Cellitti, Susan E

    2018-04-16

    Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Synthesizing labeled compounds

    International Nuclear Information System (INIS)

    London, R.E.; Matwiyoff, N.A.; Unkefer, C.J.; Walker, T.E.

    1983-01-01

    A metabolic study is presented of the chemical reactions provided by isotopic labeling and NMR spectroscopy. Synthesis of 13 C-labeled D-glucose, a 6-carbon sugar, involves adding a labeled nitrile group to the 5-carbon sugar D-arabinose by reaction with labeled hydrogen cyanide. The product of this reaction is then reduced and hydrolyzed to a mixture of the labeled sugars. The two sugars are separated by absorption chromotography. The synthesis of 13 C-labeled L-tyrosine, an amino acid, is also presented

  19. In vivo single-shot (13)C spectroscopic imaging of hyperpolarized metabolites by spatiotemporal encoding

    DEFF Research Database (Denmark)

    Schmidt, Rita; Laustsen, Christoffer; Dumez, Jean-Nicolas

    2014-01-01

    are necessary. Several approaches have been customized for hyperpolarized (13)C MRI, including CSI with a center-out k-space encoding, EPSI, and spectrally selective pulses in combination with spiral EPI acquisitions. Recent studies have described the potential of single-shot alternatives based...... temporal) data sets were obtained at 7T from a murine lymphoma tumor model....

  20. Tracing carbon flow from microphytobenthos to major bacterial groups in an intertidal marine sediment by using an in situ 13C pulse-chase method

    OpenAIRE

    Miyatake, T.; Moerdijk-Poortvliet, T.C.W.; Stal, L.J.; Boschker, H.T.S.

    2014-01-01

    Carbon flow from benthic diatoms to heterotrophic bacterial was traced in an intertidal sediment for 5 consecutive days. 13C-labeled bicarbonate was sprayed onto the sediment surface during low tide and 13C-label incorporation in major carbon pools, intermediate metabolites, and biomarkers were monitored. Phospholipid-derived fatty acid (PLFA) and ribosomal ribonucleic acid (rRNA) were used to identify the responsible members of the microbial community at class and family phylogenetic resolut...

  1. Inference of past atmospheric delta13C and P/sub CO2/ from 13C/12C measurements in tree rings

    International Nuclear Information System (INIS)

    Leavitt, S.W.

    1982-01-01

    Carbon dioxide release from fossil-fuel burning is significant enough that we may soon experience perceptible changes in climate with important human consequences. An accurate reconstruction of past 13 C/ 12 C ratios of atmospheric CO 2 may provide key constraints on the historical activity of the biosphere as CO 2 source or sink. Tree rings appear to be a repository of this information but there is much noise in the collection of previous reconstructions, presumably associated with site selection, radial variability, choice of representative wood chemical constituent, and subtle effects of climate on fractionation. This study attempts to avoid these pitfalls and develop a 50-yr delta 13 C/sub ATM/ record from juniper trees (genus Juniperus), in fact, by taking advantage of the influence of climate on fractionation. Trees were harvested from suitable sites in close proximity to weather stations with monthly records of temperature and precipitation. The most useful relationships for at most 7 of the 10 sites were delta 13 C with December temperature or precipitation, because the coefficients were nearly constant from one interval to the next and the intercepts differed. Local pollution effects are believed responsible for the three anomalous sites. The separation of these regression lines of different intervals is interpreted as the response of the trees to the changing delta 13 C of atmospheric CO 2 so that delta 13 C/sub ATM/ curves are constructed from this spacing. The shape of the best-fit reconstruction suggests the biosphere has acted as CO 2 source to about 1965 and may now be a net sink

  2. Delta /sup 13/C fractionation in Tarbela dam fish

    International Nuclear Information System (INIS)

    Latif, Z.; Sajjad, M.I.; Bilal, R.; Tasneem, M.A.; Khan, I.H.; Ali, M.

    1998-01-01

    The paper focuses on the study of naturally occurring /sup 13/C fractionation in Tarbela dam fish. Craig noted that gamma /sup 13/C values for animal tissues fall in the range as their food supply. DeNiro and Epstein demonstrated clearly that the carbon isotope composition of an animal greatly depends on its diet. The above mentioned statements were observed while studying the isotopic composition of carbon in different parts of the fish. Living fish was purchased from the Haripur side of the Tarbela lake. Different portions were separated and fish diet was collected from the fish stomach. Samples were dried in the oven at 40-50 deg. C for five days. Ground, homogenized and ignited with research grade oxygen at 900-1000 deg. C. CO and CO /sub 2/ were produced and CO was converted to CO/sub 2/ by circulation over CuO gauge furnace at 900 deg. C. CO/sub 2/ was purified using 70 deg. C slush and analyzed on Varian Mat (GD-150) mass spectrometer for gamma /sup 13/C measurements. The results show that fish flesh sup/13 C value is nearly similar to fish diet gamma /sup 13/C. gamma /sup 13/C values to different parts of the fish departed from that of the diet in the sequence: fish swim bladder (-22.04) >ribs (2-22.26)>skin (122.91)>diet (123.22)>flesh (-23.40)> vertebral column (-24.07). It is concluded that diet is easily metabolized in the fish flesh and skin tissues through blood streams without causing any pronounced fractionation. Fractionation was observed in the fish endo skeleton system due to which fish ribs become enriched in gamma /sup 13/C than vertebral column. Fractionation was also detected in visceral muscles (swim bladder) of the fish as comparison with somatic axial trunk muscle (fish flesh). (author)

  3. Label-free aptamer biosensor for selective detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Na, Weidan; Liu, Xiaotong; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2015-10-29

    We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd{sup 2+} on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results. - Highlights: • A novel strategy for the detection of thrombin was established based on BSA-CdS QDs. • DNA could serve as the co-ligands to stabilize CdS QDs and enhance the fluorescence intensity. • Thrombin could change the structure of DNA1 and quench the fluorescence of CdS QDs. • Thrombin in real sample was detected with satisfactory results.

  4. Direct observation of glycogen synthesis in human muscle with 13C NMR

    International Nuclear Information System (INIS)

    Jue, T.; Rothman, D.L.; Shulman, G.I.; Tavitian, B.A.; DeFronzo, R.A.; Shulman, R.G.

    1989-01-01

    On the basis of previous indirect measurements, skeletal muscle has been implicated as the major site of glucose uptake and it has been suggested that muscle glycogen formation is the dominant pathway. However, direct measurements of the rates of glycogen synthesis have not been possible by previous techniques. The authors have developed 13 C NMR methods to measure directly the rate of human muscle glycogen formation from infused, isotopically labeled [1- 13 C]glucose. They show that under conditions of imposed hyperglycemia and hyperinsulinemia, a majority of the infused glucose was converted to muscle glycogen in a normal man. This directly shows that muscle is the major site of glucose disposal under these conditions, and provides quantitation of the glucose flux to muscle glycogen

  5. Comparison of selective arterial spin labeling using 1D and 2D tagging RF pulses

    Energy Technology Data Exchange (ETDEWEB)

    Konstandin, Simon; Heiler, Patrick M.; Schad, Lothar R. [Heidelberg Univ., Mannheim (Germany). Computer Assisted Clinical Medicine; Scharf, Johann [Heidelberg Univ., Mannheim (Germany). Dept. of Neuroradiology

    2011-07-01

    Generic arterial spin labeling (ASL) techniques label all brain feeding arteries. In this work, we used two different selective ASL (SASL) methods to show the perfusion of one single artery. A slice selective inversion of an area including the desired vessel was compared to a multidimensional RF pulse with Gaussian profile to label only the artery of interest. Perfusion images with a resolution of 2 x 2 x 5 mm{sup 3} are shown that were acquired after tagging only the internal carotid artery of healthy volunteers. In addition, both techniques were applied to a patient with an extra-intracranial bypass to illustrate its perfusion territory. These perfusion images are consistent with a standard angiography. SASL imaging with a resolution of 2 x 2 x 5 mm{sup 3} is possible in a total scan time of 5 min. The presented MR techniques may in part replace the assessment of revascularization success by conventional angiography. (orig.)

  6. Comparison of selective arterial spin labeling using 1D and 2D tagging RF pulses

    International Nuclear Information System (INIS)

    Konstandin, Simon; Heiler, Patrick M.; Schad, Lothar R.; Scharf, Johann

    2011-01-01

    Generic arterial spin labeling (ASL) techniques label all brain feeding arteries. In this work, we used two different selective ASL (SASL) methods to show the perfusion of one single artery. A slice selective inversion of an area including the desired vessel was compared to a multidimensional RF pulse with Gaussian profile to label only the artery of interest. Perfusion images with a resolution of 2 x 2 x 5 mm 3 are shown that were acquired after tagging only the internal carotid artery of healthy volunteers. In addition, both techniques were applied to a patient with an extra-intracranial bypass to illustrate its perfusion territory. These perfusion images are consistent with a standard angiography. SASL imaging with a resolution of 2 x 2 x 5 mm 3 is possible in a total scan time of 5 min. The presented MR techniques may in part replace the assessment of revascularization success by conventional angiography. (orig.)

  7. Quantitative analysis of deuterium using the isotopic effect on quaternary {sup 13}C NMR chemical shifts

    Energy Technology Data Exchange (ETDEWEB)

    Darwish, Tamim A., E-mail: tamim.darwish@ansto.gov.au [National Deuteration Facility, Australian Nuclear Science and Technology Organisation, Locked Bag 21, Kirrawee DC, NSW 2232 (Australia); Yepuri, Nageshwar Rao; Holden, Peter J. [National Deuteration Facility, Australian Nuclear Science and Technology Organisation, Locked Bag 21, Kirrawee DC, NSW 2232 (Australia); James, Michael [Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168 (Australia)

    2016-07-13

    Quantitative analysis of specifically deuterated compounds can be achieved by a number of conventional methods, such as mass spectroscopy, or by quantifying the residual {sup 1}H NMR signals compared to signals from internal standards. However, site specific quantification using these methods becomes challenging when dealing with non-specifically or randomly deuterated compounds that are produced by metal catalyzed hydrothermal reactions in D{sub 2}O, one of the most convenient deuteration methods. In this study, deuterium-induced NMR isotope shifts of quaternary {sup 13}C resonances neighboring deuterated sites have been utilized to quantify the degree of isotope labeling of molecular sites in non-specifically deuterated molecules. By probing {sup 13}C NMR signals while decoupling both proton and deuterium nuclei, it is possible to resolve {sup 13}C resonances of the different isotopologues based on the isotopic shifts and the degree of deuteration of the carbon atoms. We demonstrate that in different isotopologues, the same quaternary carbon, neighboring partially deuterated carbon atoms, are affected to an equal extent by relaxation. Decoupling both nuclei ({sup 1}H, {sup 2}H) resolves closely separated quaternary {sup 13}C signals of the different isotopologues, and allows their accurate integration and quantification under short relaxation delays (D1 = 1 s) and hence fast accumulative spectral acquisition. We have performed a number of approaches to quantify the deuterium content at different specific sites to demonstrate a convenient and generic analysis method for use in randomly deuterated molecules, or in cases of specifically deuterated molecules where back-exchange processes may take place during work up. - Graphical abstract: The relative intensities of quaternary {sup 13}C {"1H,"2H} resonances are equal despite the different relaxation delays, allowing the relative abundance of the different deuterated isotopologues to be calculated using NMR fast

  8. Melatonin Decreases Glucose Metabolism in Prostate Cancer Cells: A 13C Stable Isotope-Resolved Metabolomic Study

    Science.gov (United States)

    Hevia, David; Gonzalez-Menendez, Pedro; Fernandez-Fernandez, Mario; Cueto, Sergio; Mayo, Juan C.

    2017-01-01

    The pineal neuroindole melatonin exerts an exceptional variety of systemic functions. Some of them are exerted through its specific membrane receptors type 1 and type 2 (MT1 and MT2) while others are mediated by receptor-independent mechanisms. A potential transport of melatonin through facilitative glucose transporters (GLUT/SLC2A) was proposed in prostate cancer cells. The prostate cells have a particular metabolism that changes during tumor progression. During the first steps of carcinogenesis, oxidative phosphorylation is reactivated while the switch to the “Warburg effect” only occurs in advanced tumors and in the metastatic stage. Here, we investigated whether melatonin might change prostate cancer cell metabolism. To do so, 13C stable isotope-resolved metabolomics in androgen sensitive LNCaP and insensitive PC-3 prostate cancer cells were employed. In addition to metabolite 13C-labeling, ATP/AMP levels, and lactate dehydrogenase or pentose phosphate pathway activity were measured. Melatonin reduces lactate labeling in androgen-sensitive cells and it also lowers 13C-labeling of tricarboxylic acid cycle metabolites and ATP production. In addition, melatonin reduces lactate 13C-labeling in androgen insensitive prostate cancer cells. Results demonstrated that melatonin limits glycolysis as well as the tricarboxylic acid cycle and pentose phosphate pathway in prostate cancer cells, suggesting that the reduction of glucose uptake is a major target of the indole in this tumor type. PMID:28933733

  9. Melatonin Decreases Glucose Metabolism in Prostate Cancer Cells: A 13C Stable Isotope-Resolved Metabolomic Study.

    Science.gov (United States)

    Hevia, David; Gonzalez-Menendez, Pedro; Fernandez-Fernandez, Mario; Cueto, Sergio; Rodriguez-Gonzalez, Pablo; Garcia-Alonso, Jose I; Mayo, Juan C; Sainz, Rosa M

    2017-07-26

    The pineal neuroindole melatonin exerts an exceptional variety of systemic functions. Some of them are exerted through its specific membrane receptors type 1 and type 2 (MT1 and MT2) while others are mediated by receptor-independent mechanisms. A potential transport of melatonin through facilitative glucose transporters (GLUT/ SLC2A ) was proposed in prostate cancer cells. The prostate cells have a particular metabolism that changes during tumor progression. During the first steps of carcinogenesis, oxidative phosphorylation is reactivated while the switch to the "Warburg effect" only occurs in advanced tumors and in the metastatic stage. Here, we investigated whether melatonin might change prostate cancer cell metabolism. To do so, 13 C stable isotope-resolved metabolomics in androgen sensitive LNCaP and insensitive PC-3 prostate cancer cells were employed. In addition to metabolite 13 C-labeling, ATP/AMP levels, and lactate dehydrogenase or pentose phosphate pathway activity were measured. Melatonin reduces lactate labeling in androgen-sensitive cells and it also lowers 13 C-labeling of tricarboxylic acid cycle metabolites and ATP production. In addition, melatonin reduces lactate 13 C-labeling in androgen insensitive prostate cancer cells. Results demonstrated that melatonin limits glycolysis as well as the tricarboxylic acid cycle and pentose phosphate pathway in prostate cancer cells, suggesting that the reduction of glucose uptake is a major target of the indole in this tumor type.

  10. Tricarboxylic acid cycle activity measured by 13C magnetic resonance spectroscopy in rats subjected to the kaolin model of obstructed hydrocephalus

    DEFF Research Database (Denmark)

    Melø, Torun M; Håberg, Asta K; Risa, Øystein

    2011-01-01

    in the amounts of glutamate, alanine and taurine. In addition, the concentration of the neuronal marker N-acetyl aspartate was decreased. (13)C Labelling of most amino acids derived from [1,6-(13)C]glucose was unchanged 2 weeks after hydrocephalus induction. The only indication of astrocyte impairment......Evaluating early changes in cerebral metabolism in hydrocephalus can help in the decision making and the timing of surgical intervention. This study was aimed at examining the tricarboxylic acid (TCA) cycle rate and (13)C label incorporation into neurotransmitter amino acids and other compounds 2...

  11. 13C mixed triglyceride breath test: a noninvasive method to assess lipase activity in children.

    Science.gov (United States)

    van Dijk-van Aalst, K; Van Den Driessche, M; van Der Schoor, S; Schiffelers, S; van't Westeinde, T; Ghoos, Y; Veereman-Wauters, G

    2001-05-01

    Results from the 13C mixed triglyceride (MTG) breath test correlate with duodenal lipase activity in adults. This noninvasive test is a potential screening and diagnostic tool for children with fat malabsorption. The aim of this study was to adapt the methodology of the MTG breath test to study test meals and sampling methods and to define normal values for healthy children of all age groups; premature and full-term infants have similar pancreatic lipase deficiencies. After parental consent was obtained, 12 premature infants ( 2 kg), 12 full-term infants (1-6 months old), 20 children (3-10 years old), and 20 teenagers (11-17 years old) were tested. All children were thriving well, had no gastrointestinal or respiratory problems, and had not received any medication that contained natural 13C. For the premature and full-term infants, a formula was prepared that had a low and stable natural 13C content mixed with 100 mg 13C-labeled MTG (1,3-distearyl, 2-[13C-carboxyl] octanoyl glycerol) and 1 g polyethylene-glycol 3350. The best accepted test meal for children over 3 years old was a slice of white bread with 5 g butter and 15 g chocolate paste, mixed with 250 mg 13C-labeled MTG, and a glass of 100 mL whole-fat milk. Children over 3 years old were able to blow through a straw in a vacutainer for collecting the breath samples. In children under 3 years old, expired air was collected by aspirating breath via a nasal prong. Carbon dioxide production was calculated according to weight, age, and sex. For healthy pediatric control participants, the mean values for cumulative excretion of 13CO2 as a percentage of the administered dose after 6 hours were 23.9 +/- 5.2% in premature infants, 31.9 +/- 7.7% in full-term infants, 32.5 +/- 5.3% in children, and 28.0 +/- 5.4% in teenagers. The mean value for healthy adults is 35.6% with a lower reference limit of 22.8%. Age-specific test meals and breath-sampling techniques for the MTG breath test were defined. The mean values for

  12. {sup 13}C relaxation in an RNA hairpin

    Energy Technology Data Exchange (ETDEWEB)

    King, G.C. [Univ. of South Wales, Kensington (Australia)]|[Rice Univ., Houston, TX (United States); Akratos, C. [Univ. of South Wales, Kensington (Australia); Xi, Z.; Michnica, M.J. [Rice Univ., Houston, TX (United States)

    1994-12-01

    This initial survey of {sup 13}C relaxation in the {triangle}TAR RNA element has generated a number of interesting results that should prove generally useful for future studies. The most readily comparable study in the literature monitored {sup 13}C relaxation of the methyl groups from unusual bases in tRNA{sup Phe}. The study, which used T{sub 1} and NOE data only, reported order parameters for the methyl group axis that ranged between 0.51 and 0.97-a range similar to that observed here. However, they reported a breakdown of the standard order parameter analysis at higher (118-MHz {sup 13}C) frequencies, which should serve to emphasize the need for a thorough exploration of suitable motional models.

  13. 13C and 31P NMR studies of myocardial metabolism

    International Nuclear Information System (INIS)

    Laughlin, M.R.

    1988-01-01

    The fluxes through two enzyme systems have been measured in perfused or in in vivo heart using NMR: phosphocreatine kinase, and glycogen synthase and phosphorylase. The rates of synthesis and degradation of glycogen were monitored in vivo in fed, fasted, and diabetic rat heart during infusions of 13 C-1-glucose and insulin using proton-decoupled 13 C-NMR at 1.9 and 4.7 tesla. The enzyme activities of glycogen synthase and glycogen phosphorylase were also measured in this tissue which had been freeze clamped at the end of the experiment, for comparison with the synthetic rates. For normal fed, fasted, and diabetic animals, synthesis rates were 0.28, 0.16, and 0.15 μmol/min.gww respectively. Glycogen synthase i activity was 0.23, 0.14, and 0.14 μmol/min.gww in these hearts at the end of the experiment, when measured at appropriate substrate and activator concentrations, and follow activation time courses that are consistent with being the main rate determinant for net synthesis in all cases. Turnover of glycogen was studied by observing the preformed 13 C-1-glycogen signal during infusion of 12 C-glucose and insulin, and was found to be close to zero. Extracted phosphorylase a activity was approximately ten times that of synthase i under these circumstances. In order to fully interpret the turnover studies, glycogenolysis of preformed 13 C-glycogen was observed after a bolus of glucagon. The glycogen had either been synthesized from 13 C-1-glucose for a single hour, or during an hour of 13 C-glucose and a subsequent hour of 12 C-glucose infusion. The author observed that breakdown follows an exponential time course related to the phosphorylase a activation state and that the last synthesized glycogen breaks down at the rate of 2.5 μmol/min.gww, five times faster than that synthesized an hour earlier

  14. 13C-nuclear magnetic resonance studies of the biosynthesis of 5-aminolevolinic acid destined for chlorophyll formation in dark-grown Scenedesmus Obliquus

    International Nuclear Information System (INIS)

    Oh-hama, Tamiko; Seto, Harvo; Miyachi, Shigetoh

    1985-01-01

    The 13 C-nuclear magnetic resonance (NMR) spectra of chlorophyll-α-formed in dark-grown Scenedesmus Obliquus (Turp.) Kutzing in the presence of (1- 13 C) glutamate, (2- 13 C) and (1- 13 C) glycine showed that the 13 C of glutamate was specifically incorporated into the eight-carbon atoms in the tetrapyrrole macrocyles derived from C-5 of 5-aminolevolinic acid (ALA), while the C-2 of glycine was only incorporated into the methyl carbon of the methoxycarbonyl group attached to the isolcyclic ring of chlorophyll a formed in the presence of (1- 13 C)-glycine. These labelling patterns provide evidence for the operation of the C 5 -pathway and against the operation of the ALA synthase pathway for chlorophyll formation in darkness. (author)

  15. 13C/12C ratios in human urine concrementes

    International Nuclear Information System (INIS)

    Hoefs, J.; Armbruster, T.

    1978-01-01

    Oxalate, uric acid, and phosphate stones have been analyzed for their carbon isotope composition. The oxalate stones show delta 13 C values between -17.0 and -19.5 pro mille, the uric acid stones between -14.9 and -19.4 pro mille, and the phosphate stones between -13.0 and -23.9 pro mille. It is proposed that endogenic rather than exogenic sources are responsible for the 13 C/ 12 C ratios of the stones. The isotopic composition of the phosphate stones seems to be influenced primarily by bacterial activity. (orig.) [de

  16. Direct 13C NMR Detection in HPLC Hyphenation Mode

    DEFF Research Database (Denmark)

    Wubshet, Sileshi Gizachew; Johansen, Kenneth; Nyberg, Nils

    2012-01-01

    Solid phase extraction (SPE) was introduced as a crucial step in the HPLC-SPE-NMR technique to enable online analyte enrichment from which proton-detected NMR experiments on submicrogram amounts from complex mixtures were possible. However, the significance of direct-detected (13)C NMR experiments...... application of HPLC-SPE-NMR analysis using direct-detected (13)C NMR spectra. HPLC column loading, accumulative SPE trappings, and the effect of different elution solvents were evaluated and optimized. A column loading of approximately 600 mug of a prefractionated triterpenoid mixture, six trappings...

  17. Analysis of neuron–astrocyte metabolic cooperation in the brain of db/db mice with cognitive decline using 13C NMR spectroscopy

    Science.gov (United States)

    Zheng, Hong; Zheng, Yongquan; Wang, Dan; Cai, Aimin; Lin, Qiuting; Zhao, Liangcai; Chen, Minjiang; Deng, Mingjie; Ye, Xinjian

    2016-01-01

    Type 2 diabetes has been linked to cognitive impairment, but its potential metabolic mechanism is still unclear. The present study aimed to explore neuron–astrocyte metabolic cooperation in the brain of diabetic (db/db, BKS.Cg-m+/+ Leprdb/J) mice with cognitive decline using 13C NMR technique in combination with intravenous [2-13C]-acetate and [3-13C]-lactate infusions. We found that the 13C-enrichment from [2-13C]-acetate into tricarboxylic acid cycle intermediate, succinate, was significantly decreased in db/db mice with cognitive decline compared with wild-type (WT, C57BLKS/J) mice, while an opposite result was obtained after [3-13C]-lactate infusion. Relative to WT mice, db/db mice with cognitive decline had significantly lower 13C labeling percentages in neurotransmitters including glutamine, glutamate, and γ-aminobutyric acid after [2-13C]-acetate infusion. However, [3-13C]-lactate resulted in increased 13C-enrichments in neurotransmitters in db/db mice with cognitive decline. This may indicate that the disturbance of neurotransmitter metabolism occurred during the development of cognitive decline. In addition, a reduction in 13C-labeling of lactate and an increase in gluconeogenesis were found from both labeled infusions in db/db mice with cognitive decline. Therefore, our results suggest that the development of cognitive decline in type 2 diabetes may be implicated to an unbalanced metabolism in neuron–astrocyte cooperation and an enhancement of gluconeogenesis. PMID:26762505

  18. Analysis of neuron-astrocyte metabolic cooperation in the brain of db/db mice with cognitive decline using 13C NMR spectroscopy.

    Science.gov (United States)

    Zheng, Hong; Zheng, Yongquan; Wang, Dan; Cai, Aimin; Lin, Qiuting; Zhao, Liangcai; Chen, Minjiang; Deng, Mingjie; Ye, Xinjian; Gao, Hongchang

    2017-01-01

    Type 2 diabetes has been linked to cognitive impairment, but its potential metabolic mechanism is still unclear. The present study aimed to explore neuron-astrocyte metabolic cooperation in the brain of diabetic (db/db, BKS.Cg-m +/+ Leprdb/J) mice with cognitive decline using 13 C NMR technique in combination with intravenous [2- 13 C]-acetate and [3- 13 C]-lactate infusions. We found that the 13 C-enrichment from [2- 13 C]-acetate into tricarboxylic acid cycle intermediate, succinate, was significantly decreased in db/db mice with cognitive decline compared with wild-type (WT, C57BLKS/J) mice, while an opposite result was obtained after [3- 13 C]-lactate infusion. Relative to WT mice, db/db mice with cognitive decline had significantly lower 13 C labeling percentages in neurotransmitters including glutamine, glutamate, and γ-aminobutyric acid after [2- 13 C]-acetate infusion. However, [3- 13 C]-lactate resulted in increased 13 C-enrichments in neurotransmitters in db/db mice with cognitive decline. This may indicate that the disturbance of neurotransmitter metabolism occurred during the development of cognitive decline. In addition, a reduction in 13 C-labeling of lactate and an increase in gluconeogenesis were found from both labeled infusions in db/db mice with cognitive decline. Therefore, our results suggest that the development of cognitive decline in type 2 diabetes may be implicated to an unbalanced metabolism in neuron-astrocyte cooperation and an enhancement of gluconeogenesis. © The Author(s) 2016.

  19. Two-Scale 13C Metabolic Flux Analysis for Metabolic Engineering.

    Science.gov (United States)

    Ando, David; Garcia Martin, Hector

    2018-01-01

    Accelerating the Design-Build-Test-Learn (DBTL) cycle in synthetic biology is critical to achieving rapid and facile bioengineering of organisms for the production of, e.g., biofuels and other chemicals. The Learn phase involves using data obtained from the Test phase to inform the next Design phase. As part of the Learn phase, mathematical models of metabolic fluxes give a mechanistic level of comprehension to cellular metabolism, isolating the principle drivers of metabolic behavior from the peripheral ones, and directing future experimental designs and engineering methodologies. Furthermore, the measurement of intracellular metabolic fluxes is specifically noteworthy as providing a rapid and easy-to-understand picture of how carbon and energy flow throughout the cell. Here, we present a detailed guide to performing metabolic flux analysis in the Learn phase of the DBTL cycle, where we show how one can take the isotope labeling data from a 13 C labeling experiment and immediately turn it into a determination of cellular fluxes that points in the direction of genetic engineering strategies that will advance the metabolic engineering process.For our modeling purposes we use the Joint BioEnergy Institute (JBEI) Quantitative Metabolic Modeling (jQMM) library, which provides an open-source, python-based framework for modeling internal metabolic fluxes and making actionable predictions on how to modify cellular metabolism for specific bioengineering goals. It presents a complete toolbox for performing different types of flux analysis such as Flux Balance Analysis, 13 C Metabolic Flux Analysis, and it introduces the capability to use 13 C labeling experimental data to constrain comprehensive genome-scale models through a technique called two-scale 13 C Metabolic Flux Analysis (2S- 13 C MFA) [1]. In addition to several other capabilities, the jQMM is also able to predict the effects of knockouts using the MoMA and ROOM methodologies. The use of the jQMM library is

  20. Natural isotope correction of MS/MS measurements for metabolomics and (13)C fluxomics.

    Science.gov (United States)

    Niedenführ, Sebastian; ten Pierick, Angela; van Dam, Patricia T N; Suarez-Mendez, Camilo A; Nöh, Katharina; Wahl, S Aljoscha

    2016-05-01

    Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of (13)C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full use of LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; (13)C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation. © 2015 Wiley Periodicals, Inc.

  1. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

    International Nuclear Information System (INIS)

    Kirley, Terence L.; Greis, Kenneth D.; Norman, Andrew B.

    2016-01-01

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’) 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’) 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be used, simplifying

  2. Internal motion time scales of a small, highly stable and disulfide-rich protein: A 15N, 13C NMR and molecular dynamics study

    International Nuclear Information System (INIS)

    Guenneugues, Marc; Gilquin, Bernard; Wolff, Nicolas; Menez, Andre; Zinn-Justin, Sophie

    1999-01-01

    Motions of the backbone CαHα and threonine CβHβ bonds of toxin α were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H → 13C NOE were obtained, as well as the variations of R1ρ(90 deg.) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several CαHα and threonine CβHβ experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin α, a highly stable protein (Tm=75 deg. C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 μs to 10 ms

  3. Quantification of Lignin and Its Structural Features in Plant Biomass Using 13C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS.

    Science.gov (United States)

    van Erven, Gijs; de Visser, Ries; Merkx, Donny W H; Strolenberg, Willem; de Gijsel, Peter; Gruppen, Harry; Kabel, Mirjam A

    2017-10-17

    Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13 C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12 C and 13 C lignin were isolated from nonlabeled and uniformly 13 C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13 C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12 C lignin analogue and was shown to be extremely accurate (>99.9%, R 2 > 0.999) and precise (RSD corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.

  4. Revisiting the metathesis of 13C-monolabeled ethane

    KAUST Repository

    Maury, Olivier

    2010-12-13

    The metathesis of 13C-monolabeled ethane leads to the parallel occurrence of degenerate and productive reactions, affording the statistical distribution of the various product isotopomers, which can be rationalized in terms of a mechanistic reaction scheme combining both processes. © 2010 American Chemical Society.

  5. 13C-NMR spectra and bonding situation in ketenimines

    International Nuclear Information System (INIS)

    Firl, J.; Runge, W.; Hartmann, W.; Utikal, H.P.

    1975-01-01

    13 C-NMR spectra of a series of substituted ketenimines are reported. The terminal carbon resonances are found at unusual high fields between delta 37 and 78, while the central carbon signals appear around delta 189 - 196. On the basis of these results, the bonding situation in ketenimines has been discussed. (auth.)

  6. δ13C-CH4 in ice core samples

    DEFF Research Database (Denmark)

    Sperlich, Peter

    Ice core records of δ13C-CH4 reflect the variability of CH4 biogeochemistry in response to climate change and show this system is far more complex than expected. The first part of this work is concerned with the development of analytical techniques that allow 1) precise referencing and 2) measure......Ice core records of δ13C-CH4 reflect the variability of CH4 biogeochemistry in response to climate change and show this system is far more complex than expected. The first part of this work is concerned with the development of analytical techniques that allow 1) precise referencing and 2......) measurements of δ13C-CH4 in ice core samples as is required when δ13C-CH4 records that are measured in several laboratories are merged for analysis. Both the referencing and measurement techniques have been compared to further laboratories which proofed the accuracy of the analytical systems. The second part...

  7. A new, 13C-based material for neutron targets

    International Nuclear Information System (INIS)

    Romanenko, A.I.; Anikeeva, O.B.; Gorbachev, R.V.; Zhmurikov, E.I.; Gubin, K.V.; Logachev, P.V.; Avilov, M.S.; Tsybulya, S.V.; Kryukova, G.N.; Burgina, E.B.; Tecchio, L.

    2005-01-01

    A 13 C-based neutron-target material is investigated using X-ray diffraction, IR absorption and Raman scattering spectroscopies, transmission electron microscopy, and electrical (conductivity, magnetoresistance, and Hall effect) measurements before and after high-power electron irradiation for various lengths of time [ru

  8. In vivo 13C MRS in the mouse brain at 14.1 Tesla and metabolic flux quantification under infusion of [1,6-13C2]glucose.

    Science.gov (United States)

    Lai, Marta; Lanz, Bernard; Poitry-Yamate, Carole; Romero, Jackeline F; Berset, Corina M; Cudalbu, Cristina; Gruetter, Rolf

    2017-01-01

    In vivo 13 C magnetic resonance spectroscopy (MRS) enables the investigation of cerebral metabolic compartmentation while, e.g. infusing 13 C-labeled glucose. Metabolic flux analysis of 13 C turnover previously yielded quantitative information of glutamate and glutamine metabolism in humans and rats, while the application to in vivo mouse brain remains exceedingly challenging. In the present study, 13 C direct detection at 14.1 T provided highly resolved in vivo spectra of the mouse brain while infusing [1,6- 13 C 2 ]glucose for up to 5 h. 13 C incorporation to glutamate and glutamine C4, C3, and C2 and aspartate C3 were detected dynamically and fitted to a two-compartment model: flux estimation of neuron-glial metabolism included tricarboxylic acid cycle (TCA) flux in astrocytes (V g  = 0.16 ± 0.03 µmol/g/min) and neurons (V TCA n  = 0.56 ± 0.03 µmol/g/min), pyruvate carboxylase activity (V PC  = 0.041 ± 0.003 µmol/g/min) and neurotransmission rate (V NT  = 0.084 ± 0.008 µmol/g/min), resulting in a cerebral metabolic rate of glucose (CMR glc ) of 0.38 ± 0.02 µmol/g/min, in excellent agreement with that determined with concomitant 18 F-fluorodeoxyglucose positron emission tomography ( 18 FDG PET).We conclude that modeling of neuron-glial metabolism in vivo is accessible in the mouse brain from 13 C direct detection with an unprecedented spatial resolution under [1,6- 13 C 2 ]glucose infusion.

  9. Studies of the pH dependence of 13C shifts and carbon-carbon coupling constants of [U-13C]aspartic and -glutamic acids

    International Nuclear Information System (INIS)

    London, R.E.; Walker, T.E.; Kollman, V.H.; Matwiyoff, N.A.

    1978-01-01

    13 C NMR studies of the chemical shifts and carbon--carbon spin--spin coupling constants of 90% [U- 13 C]aspartic and -glutamic acids are reported. Effects of titration of the two carboxyl groups are separated computationally and the results compared with those for asparagine and glutamine, aspartate and glutamate containing peptides, and a series of amino-n-butyric acids. The results indicate that the carboxyl carbon shift resulting from titration of the carboxyl group is strongly dependent on its distance (number of bonds) from an amino group. Alternatively, remote methyl groups exhibit a much smaller titration induced shift than carboxyl groups in the corresponding position. Significant remote effects of pH titration on the one-bond carbon-carbon coupling are also observed, particularly for couplings involving the side-chain carboxyl carbons. These results are discussed in terms of polarization of the C--O bonds in response to titration of a remote carboxyl group. Values of 3 J/sub CC/ in asparate and glutamate indicate a strong conformational dependence. Rotamer populations predicted on the basis of the observed couplings and theoretical INDO calculations are in good agreement with values based on analysis of the 3 J/sub HH/ and 3 J/sub CH/ couplings. For a given conformation of glutamic acid, it is found that 3 J 14 is considerably smaller than 3 J 25 . This result is consistent with obsrvations on a number of other 13 C-labeled amino acids. 5 figures, 4 tables

  10. Optical hyperpolarization of 13C nuclear spins in nanodiamond ensembles

    Science.gov (United States)

    Chen, Q.; Schwarz, I.; Jelezko, F.; Retzker, A.; Plenio, M. B.

    2015-11-01

    Dynamical nuclear polarization holds the key for orders of magnitude enhancements of nuclear magnetic resonance signals which, in turn, would enable a wide range of novel applications in biomedical sciences. However, current implementations of DNP require cryogenic temperatures and long times for achieving high polarization. Here we propose and analyze in detail protocols that can achieve rapid hyperpolarization of 13C nuclear spins in randomly oriented ensembles of nanodiamonds at room temperature. Our protocols exploit a combination of optical polarization of electron spins in nitrogen-vacancy centers and the transfer of this polarization to 13C nuclei by means of microwave control to overcome the severe challenges that are posed by the random orientation of the nanodiamonds and their nitrogen-vacancy centers. Specifically, these random orientations result in exceedingly large energy variations of the electron spin levels that render the polarization and coherent control of the nitrogen-vacancy center electron spins as well as the control of their coherent interaction with the surrounding 13C nuclear spins highly inefficient. We address these challenges by a combination of an off-resonant microwave double resonance scheme in conjunction with a realization of the integrated solid effect which, together with adiabatic rotations of external magnetic fields or rotations of nanodiamonds, leads to a protocol that achieves high levels of hyperpolarization of the entire nuclear-spin bath in a randomly oriented ensemble of nanodiamonds even at room temperature. This hyperpolarization together with the long nuclear-spin polarization lifetimes in nanodiamonds and the relatively high density of 13C nuclei has the potential to result in a major signal enhancement in 13C nuclear magnetic resonance imaging and suggests functionalized and hyperpolarized nanodiamonds as a unique probe for molecular imaging both in vitro and in vivo.

  11. Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

    Directory of Open Access Journals (Sweden)

    Shingo Sotoma

    2016-03-01

    Full Text Available The impeccable photostability of fluorescent nanodiamonds (FNDs is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

  12. Effects of pentylenetetrazole and glutamate on metabolism of [U-(13)C]glucose in cultured cerebellar granule neurons.

    Science.gov (United States)

    Eloqayli, Haytham; Qu, Hong; Unsgård, Geirmund; Sletvold, Olav; Hadidi, Hakam; Sonnewald, Ursula

    2002-02-01

    This study was performed to analyze the effects of glutamate and the epileptogenic agent pentylenetetrazole (PTZ) on neuronal glucose metabolism. Cerebellar granule neurons were incubated for 2 h in medium containing 3 mM [U-(13)C]glucose, with and without 0.25 mM glutamate and/or 10 mM PTZ. In the presence of PTZ, decreased glucose consumption with unchanged lactate release was observed, indicating decreased glucose oxidation. PTZ also slowed down tricarboxylic acid (TCA) cycle activity as evidenced by the decreased amounts of labeled aspartate and [1,2-(13)C]glutamate. When glutamate was present, glucose consumption was also decreased. However, the amount of glutamate, derived from [U-(13)C]glucose via the first turn of the TCA cycle, was increased. The decreased amount of [1,2-(13)C]glutamate, derived from the second turn in the TCA cycle, and increased amount of aspartate indicated the dilution of label due to the entrance of unlabeled glutamate into TCA cycle. In the presence of glutamate plus PTZ, the effect of PTZ was enhanced by glutamate. Labeled alanine was detected only in the presence of glutamate plus PTZ, which indicated that oxaloacetate was a better amino acid acceptor than pyruvate. Furthermore, there was also evidence for intracellular compartmentation of oxaloacetate metabolism. Glutamate and PTZ caused similar metabolic changes, however, via different mechanisms. Glutamate substituted for glucose as energy substrate in the TCA cycle, whereas, PTZ appeared to decrease mitochondrial activity.

  13. SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis

    Science.gov (United States)

    Kogadeeva, Maria

    2016-01-01

    Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses. PMID:27626798

  14. Automated selected reaction monitoring software for accurate label-free protein quantification.

    Science.gov (United States)

    Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

    2012-07-06

    Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

  15. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

    Science.gov (United States)

    Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

    2016-11-25

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI.

    Science.gov (United States)

    Xu, Tao; Mayer, Dirk; Gu, Meng; Yen, Yi-Fen; Josan, Sonal; Tropp, James; Pfefferbaum, Adolf; Hurd, Ralph; Spielman, Daniel

    2011-10-01

    With signal-to-noise ratio enhancements on the order of 10,000-fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1-(13)C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1-(13)C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time-to-peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small-flip-angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral-based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo (13)C labeling of lactate and alanine after a bolus injection of [1-(13)C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis-Menten-like formulation, with the resulting estimated apparent maximal reaction velocity V(max) and apparent Michaelis constant K(M) being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis-Menten kinetics, it is conceptually different. In this study, we focus on the (13)C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport

  17. Sequential nearest-neighbor effects on computed {sup 13}C{sup {alpha}} chemical shifts

    Energy Technology Data Exchange (ETDEWEB)

    Vila, Jorge A. [Cornell University, Baker Laboratory of Chemistry and Chemical Biology (United States); Serrano, Pedro; Wuethrich, Kurt [The Scripps Research Institute, Department of Molecular Biology (United States); Scheraga, Harold A., E-mail: has5@cornell.ed [Cornell University, Baker Laboratory of Chemistry and Chemical Biology (United States)

    2010-09-15

    To evaluate sequential nearest-neighbor effects on quantum-chemical calculations of {sup 13}C{sup {alpha}} chemical shifts, we selected the structure of the nucleic acid binding (NAB) protein from the SARS coronavirus determined by NMR in solution (PDB id 2K87). NAB is a 116-residue {alpha}/{beta} protein, which contains 9 prolines and has 50% of its residues located in loops and turns. Overall, the results presented here show that sizeable nearest-neighbor effects are seen only for residues preceding proline, where Pro introduces an overestimation, on average, of 1.73 ppm in the computed {sup 13}C{sup {alpha}} chemical shifts. A new ensemble of 20 conformers representing the NMR structure of the NAB, which was calculated with an input containing backbone torsion angle constraints derived from the theoretical {sup 13}C{sup {alpha}} chemical shifts as supplementary data to the NOE distance constraints, exhibits very similar topology and comparable agreement with the NOE constraints as the published NMR structure. However, the two structures differ in the patterns of differences between observed and computed {sup 13}C{sup {alpha}} chemical shifts, {Delta}{sub ca,i}, for the individual residues along the sequence. This indicates that the {Delta}{sub ca,i} -values for the NAB protein are primarily a consequence of the limited sampling by the bundles of 20 conformers used, as in common practice, to represent the two NMR structures, rather than of local flaws in the structures.

  18. OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Nielsen Lars K

    2009-05-01

    Full Text Available Abstract Background The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i tracer cultivation on 13C substrates, (ii 13C labelling analysis by mass spectrometry and (iii mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis. Results We have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing it's user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly ( Conclusion We have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and

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

    Science.gov (United States)

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

    2016-04-01

    Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

  20. Deconvolution of the tree ring based delta13C record

    International Nuclear Information System (INIS)

    Peng, T.; Broecker, W.S.; Freyer, H.D.; Trumbore, S.

    1983-01-01

    We assumed that the tree-ring based 13 C/ 12 C record constructed by Freyer and Belacy (1983) to be representative of the fossil fuel and forest-soil induced 13 C/ 12 C change for atmospheric CO 2 . Through the use of a modification of the Oeschger et al. ocean model, we have computed the contribution of the combustion of coal, oil, and natural gas to this observed 13 C/ 12 C change. A large residual remains when the tree-ring-based record is corrected for the contribution of fossil fuel CO 2 . A deconvolution was performed on this residual to determine the time history and magnitude of the forest-soil reservoir changes over the past 150 years. Several important conclusions were reached. (1) The magnitude of the integrated CO 2 input from these sources was about 1.6 times that from fossil fuels. (2) The forest-soil contribution reached a broad maximum centered at about 1900. (3) Over the 2 decade period covered by the Mauna Loa atmospheric CO 2 content record, the input from forests and soils was about 30% that from fossil fuels. (4) The 13 C/ 12 C trend over the last 20 years was dominated by the input of fossil fuel CO 2 . (5) The forest-soil release did not contribute significantly to the secular increase in atmospheric CO 2 observed over the last 20 years. (6) The pre-1850 atmospheric p2 values must have been in the range 245 to 270 x 10 -6 atmospheres

  1. Electronic structure and physical properties of 13C carbon composite

    OpenAIRE

    Zhmurikov, Evgenij

    2015-01-01

    This review is devoted to the application of graphite and graphite composites in science and technology. Structure and electrical properties, as so technological aspects of producing of high-strength artificial graphite and dynamics of its destruction are considered. These type of graphite are traditionally used in the nuclear industry. Author was focused on the properties of graphite composites based on carbon isotope 13C. Generally, the review relies on the original results and concentrates...

  2. EDGE2D Simulations of JET 13C Migration Experiments

    International Nuclear Information System (INIS)

    Strachan, J.D.; Coad, J.P.; Corrigan, G.; Matthews, G.F.; Spence, J.

    2004-01-01

    Material migration has received renewed interest due to tritium retention associated with carbon transport to remote vessel locations. Those results influence the desirability of carbon usage on ITER. Subsequently, additional experiments have been performed, including tracer experiments attempting to identify material migration from specific locations. In this paper, EDGE2D models a well-diagnosed JET 13 C tracer migration experiment. The role of SOL flows upon the migration patterns is identified

  3. The synthesis of tritium, carbon-14 and stable isotope labelled selective estrogen receptor degraders.

    Science.gov (United States)

    Bragg, Ryan A; Bushby, Nick; Ericsson, Cecilia; Kingston, Lee P; Ji, Hailong; Elmore, Charles S

    2016-09-01

    As part of a Medicinal Chemistry program aimed at developing an orally bioavailable selective estrogen receptor degrader, a number of tritium, carbon-14, and stable isotope labelled (E)-3-[4-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl]prop-2-enoic acids were required. This paper discusses 5 synthetic approaches to this compound class. Copyright © 2016 John Wiley & Sons, Ltd.

  4. Spin-locking and cross-polarization under magic-angle spinning of uniformly labeled solids.

    Science.gov (United States)

    Hung, Ivan; Gan, Zhehong

    2015-07-01

    Spin-locking and cross-polarization under magic-angle spinning are investigated for uniformly (13)C and (15)N labeled solids. In particular, the interferences from chemical shift anisotropy, and (1)H heteronuclear and (13)C homonuclear dipolar couplings are identified. The physical origin of these interferences provides guidelines for selecting the best (13)C and (15)N polarization transfer rf fields. Optimal settings for both the zero- and double-quantum cross-polarization transfer mechanisms are recommended. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. PEDOGENIC CARBONATE δ13C AND ENVIRONMENTAL PRECIPITATION CONDITIONS

    Directory of Open Access Journals (Sweden)

    Marcella Catoni

    2011-12-01

    Full Text Available Carbon isotopic analysis is a useful tool for investigating paleoenvironments, as the pedogenic carbonate δ13C is related to δ13CSOM and to the proportions of C3/C4 plants. In this work we interpreted the paleoenvironmental conditions at the time of carbonate precipitation in soils formed under different climates and during different geological ages. Samples were taken from a Bk (PR1, Holocene and from two Bkm horizons (PR2 and PR3, Pleistocene. When the mean δ13C plant values and the most plausible paleotemperatures were used in the evaluation, PR1 showed a lower percentage of C4 plants (48% than Pleistocene soils (~53%, in agreement with paleoclimate changes. When instead the δ13C values of current plants were used for PR1, C4 plants ranged from 59 (12°C to 66% (18°C, suggesting two possible interpretations: either plant species changed during the Holocene, or the plant mean values normally used in the literature are not suitable for Pleistocene reconstructions

  6. Galactose oxidation using {sup 13}C in healthy and galactosemic children

    Energy Technology Data Exchange (ETDEWEB)

    Resende-Campanholi, D.R. [Departamento de Puericultura e Pediatria, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Porta, G. [Unidade de Hepatologia Pediátrica, Instituto da Criança Prof. Pedro de Alcântara, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Ferrioli, E.; Pfrimer, K. [Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ciampo, L.A. Del; Junior, J.S. Camelo [Departamento de Puericultura e Pediatria, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2015-01-20

    Galactosemia is an inborn error of galactose metabolism that occurs mainly as the outcome of galactose-1-phosphate uridyltransferase (GALT) deficiency. The ability to assess galactose oxidation following administration of a galactose-labeled isotope (1-{sup 13}C-galactose) allows the determination of galactose metabolism in a practical manner. We aimed to assess the level of galactose oxidation in both healthy and galactosemic Brazilian children. Twenty-one healthy children and seven children with galactosemia ranging from 1 to 7 years of age were studied. A breath test was used to quantitate {sup 13}CO{sub 2} enrichment in exhaled air before and at 30, 60, and 120 min after the oral administration of 7 mg/kg of an aqueous solution of 1-{sup 13}C-galactose to all children. The molar ratios of {sup 13}CO{sub 2} and {sup 12}CO{sub 2} were quantified by the mass/charge ratio (m/z) of stable isotopes in each air sample by gas-isotope-ratio mass spectrometry. In sick children, the cumulative percentage of {sup 13}C from labeled galactose (CUMPCD) in the exhaled air ranged from 0.03% at 30 min to 1.67% at 120 min. In contrast, healthy subjects showed a much broader range in CUMPCD, with values from 0.4% at 30 min to 5.58% at 120 min. The study found a significant difference in galactose oxidation between children with and without galactosemia, demonstrating that the breath test is useful in discriminating children with GALT deficiencies.

  7. Probing cardiac metabolism by hyperpolarized 13C MR using an exclusively endogenous substrate mixture and photo-induced nonpersistent radicals

    DEFF Research Database (Denmark)

    Bastiaansen, Jessica A M; Yoshihara, Hikari A I; Capozzi, Andrea

    2018-01-01

    dissolved, and the radical-free hyperpolarized solution was rapidly transferred into an injection pump located inside a 9.4T scanner. The hyperpolarized solution was injected in healthy rats to measure cardiac metabolism in vivo. Ultraviolet irradiation created nonpersistent radicals in a mixture containing......To probe the cardiac metabolism of carbohydrates and short chain fatty acids simultaneously in vivo following the injection of a hyperpolarized 13 C-labeled substrate mixture prepared using photo-induced nonpersistent radicals. Droplets of mixed [1-13 C]pyruvic and [1-13 C]butyric acids were frozen...... into glassy beads in liquid nitrogen. Ethanol addition was investigated as a means to increase the polarization level. The beads were irradiated with ultraviolet light and the radical concentration was measured by ESR spectroscopy. Following dynamic nuclear polarization in a 7T polarizer, the beads were...

  8. Radioiodinated methylene blue for melanoma targeting: Chemical characterisation and tumour selectivity of labelled components

    International Nuclear Information System (INIS)

    Blower, Philip J.; Clark, Katherine; Link, Eva M.

    1997-01-01

    Radioiodinated methylene blue contains a mixture of components showing selective uptake in human pigmented melanoma, and it has potential for imaging and therapy. Nuclear magnetic resonance and mass spectroscopic studies show that the majority of the radioactivity (85%) is in the form of monoiodinated methylene blue, 4-iodo-3-methylamino-7-dimethylaminophenaza thionium chloride. The amino group ortho-to iodine has become demethylated to a mono-methylamino group. The remainder (15%) of the mixture is the doubly labelled 4,5-diiodo-3,7-bis(methylamino) phenazathionium chloride. The separated components show similar tumour selectivity in athymic mice bearing human pigmented melanomas

  9. Synthesis of 14C- and 3H-labeled fluoxetine, a selective serotonin uptake inhibitor

    International Nuclear Information System (INIS)

    Robertson, D.W.; Krushinski, J.H.; Wong, D.T.; Kau, D.

    1987-01-01

    Fluoxetine (N-methyl-γ-(4-(trifluoromethyl)phenoxy) benzenepropanamine) is a potent, highly selective serotonin uptake inhibitor that is useful in treating a variety of major psychiatric derangements. We have synthesized this compound in 14 C- and 3 H-labeled forms. The tritium label was introduced in the final step by catalytic dehalogenation of the brominated fluoxetine precursor. Reaction conditions could be controlled such that catalytic hydrogenolysis of the labile C-O benzylic bond was minimized. Following HPLC purification, [ 3 H]-fluoxetine was obtained in a state of high radiochemical purity (98%) and specific activity (20.4 Ci/mmol). The 14 C-label was introduced in the final step via a nucleophilic aromatic substitution reaction between the sodium salt of α-(2-(methylamino)ethyl)benzenemethanol and uniformly ring-labeled p-chlorobenzotrifluoride. Following purification by flash chromatography, [ 14 C]-fluoxetine was obtained in 98.3% radiochemical purity with a specific activity of 5.52 mCi/mmol. (author)

  10. The effect of energy and traffic light labelling on parent and child fast food selection: a randomised controlled trial.

    Science.gov (United States)

    Dodds, Pennie; Wolfenden, Luke; Chapman, Kathy; Wellard, Lyndal; Hughes, Clare; Wiggers, John

    2014-02-01

    Labelling of food from fast food restaurants at point-of-purchase has been suggested as one strategy to reduce population energy consumption and contribute to reductions in obesity prevalence. The aim of this study was to examine the effects of energy and single traffic light labelling systems on the energy content of child and adult intended food purchases. The study employed a randomised controlled trial design. English speaking parents of children aged between three and 12 years were recruited from an existing research cohort. Participants were mailed one of three hypothetical fast food menus. Menus differed in their labelling technique – either energy labels, single traffic light labels, or a no-label control. Participants then completed a telephone survey which assessed intended food purchases for both adult and child. The primary trial outcome was total energy of intended food purchase. A total of 329 participants completed the follow-up telephone interview. Eighty-two percent of the energy labelling group and 96% of the single traffic light labelling group reported noticing labelling information on their menu. There were no significant differences in total energy of intended purchases of parents, or intended purchases made by parents for children, between the menu labelling groups, or between menu labelling groups by socio-demographic subgroups. This study provided no evidence to suggest that energy labelling or single traffic light labelling alone were effective in reducing the energy of fast food items selected from hypothetical fast food menus for purchase. Additional complementary public health initiatives promoting the consumption of healthier foods identified by labelling, and which target other key drivers of menu item selection in this setting may be required.

  11. Constraining Genome-Scale Models to Represent the Bow Tie Structure of Metabolism for 13C Metabolic Flux Analysis

    Directory of Open Access Journals (Sweden)

    Tyler W. H. Backman

    2018-01-01

    Full Text Available Determination of internal metabolic fluxes is crucial for fundamental and applied biology because they map how carbon and electrons flow through metabolism to enable cell function. 13 C Metabolic Flux Analysis ( 13 C MFA and Two-Scale 13 C Metabolic Flux Analysis (2S- 13 C MFA are two techniques used to determine such fluxes. Both operate on the simplifying approximation that metabolic flux from peripheral metabolism into central “core” carbon metabolism is minimal, and can be omitted when modeling isotopic labeling in core metabolism. The validity of this “two-scale” or “bow tie” approximation is supported both by the ability to accurately model experimental isotopic labeling data, and by experimentally verified metabolic engineering predictions using these methods. However, the boundaries of core metabolism that satisfy this approximation can vary across species, and across cell culture conditions. Here, we present a set of algorithms that (1 systematically calculate flux bounds for any specified “core” of a genome-scale model so as to satisfy the bow tie approximation and (2 automatically identify an updated set of core reactions that can satisfy this approximation more efficiently. First, we leverage linear programming to simultaneously identify the lowest fluxes from peripheral metabolism into core metabolism compatible with the observed growth rate and extracellular metabolite exchange fluxes. Second, we use Simulated Annealing to identify an updated set of core reactions that allow for a minimum of fluxes into core metabolism to satisfy these experimental constraints. Together, these methods accelerate and automate the identification of a biologically reasonable set of core reactions for use with 13 C MFA or 2S- 13 C MFA, as well as provide for a substantially lower set of flux bounds for fluxes into the core as compared with previous methods. We provide an open source Python implementation of these algorithms at https://github.com/JBEI/limitfluxtocore.

  12. Biosynthesis of quinolizidine alkaloids. Incorporation of [1-amino-15N, 1-13C] cadaverine into lupanine, 13-hydroxylupanine, and angustifoline

    International Nuclear Information System (INIS)

    Rana, J.; Robins, D.J.

    1985-01-01

    The labelling patterns in (+)-lupanine, (+)-13-hydroxylupanine, and (+)-angustifoline derived biosynthetically from [1-amino- 15 N,1- 13 C]-1,5-diaminopentane (cadaverine) have been established by 13 C n.m.r. spectroscopy. Three cadaverine units are incorporated to about the same extent into each of these three alkaloids. The presence of two doublets due to 13 C- 15 N coupling in the 13 C brace 1 H brace n.m.r. spectra associated with C-2 and C-15 of lupanine and 13-hydroxylupanine, and one 13 C- 15 N doublet at C-2 of angustifoline, indicate that two of the cadaverine units are converted into the outer rings of the tetracyclic quinolizidine alkaloids in a specific fashion. (author)

  13. Trends in stomatal density and 13C/12C ratios of Pinus flexilis needles during last glacial-interglacial cycle

    International Nuclear Information System (INIS)

    Van de Water, P.K.; Leavitt, S.W.; Betancourt, J.L.

    1994-01-01

    Measurements of stomatal density and delta 13C of limber pine (Pinus flexilis) needles (leaves) preserved in pack rat middens from the Great Basin reveal shifts in plant physiology and leaf morphology during the last 30,000 years. Sites were selected so as to offset glacia to Holocene climatic differences and thus to isolate the effects of changing atmospheric CO2 levels. Stomatal density decreased approximately 17 percent and delta 13C decreased approximately 1.5 per ml during deglaciation from 15,000 to 12,000 years ago, concomitant with a 30 percent increase in atmospheric CO2. Water-use efficiency increased approximately 15 percent during deglaciation, if temperature and humidity were held constant and the proxy values for CO2 and delta 13C of past atmospheres are accurate. The delta 13C variations may help constrain hypotheses about the redistribution of carbon between the atmosphere and biosphere during the last glacial-interglacial cycle

  14. Application of 15N labeling to topics in molecular hematology

    International Nuclear Information System (INIS)

    Lapidot, A.; Irving, C.S.

    1975-01-01

    The amount of information which can be obtained from many types of spectrometric analysis of compounds of hematological interest can be greatly enhanced when measurements are made on a series of isotopically labeled compounds. A murine Friend virus-induced erythroleukemic cell (FLC) culture was found to be a superior biosynthetic system for the preparation of highly and selectively 15 N and 13 C enriched hemoglobins. A mutant of Rhodopseudomonas spheroides was found suitable for the preparation of larger quantities of >90 percent enriched protoporphyrin-IX- 15 N and coproporphyrin-III-- 15 N. A comparison of the 15 N and 13 C NMR spectra of FLC carbomonoxy-[Gly- 15 N]-hemoglobin, carbomonoxy-[Gly- 13 C/sub alpha/]-hemoglobin, α and β globin-[Gly- 15 N] and globin-[Gly- 13 C/sub alpha/] demonstrated 1) 15 N peptide chemical shifts are sensitive to polypeptide sequence, whereas 13 C α-carbon chemical shifts are not, (2) variations in the solvation of the peptide N-H group can be detected in the 15 N spectra but not the 13 C spectra, (3) 15 N heme resonances could not be detected, whereas 13 C resonances could. These studies indicated that in hemoglobin the glycyl N-H resonances are either solvated by H 2 O or hydrogen bonded to peptide C=0 groups. In denatured globin, the majority of the glycyl residues are rapidly exchanging between these two states

  15. Measurement of δ13C values of soil amino acids by GC-C-IRMS using trimethylsilylation: a critical assessment.

    Science.gov (United States)

    Rubino, Mauro; Milin, Sylvie; D'Onofrio, Antonio; Signoret, Patrick; Hatté, Christine; Balesdent, Jérôme

    2014-01-01

    In this study, we evaluated trimethylsilyl (TMS) derivatives as derivatization reagents for the compound-specific stable carbon isotope analysis of soil amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We used non-proteinogenic amino acids to show that the extraction-derivatization-analysis procedure provides a reliable method to measure δ(13)C values of amino acids extracted from soil. However, we found a number of drawbacks that significantly increase the final total uncertainty. These include the following: production of multiple peaks for each amino acid, identified as di-, tri- and tetra-TMS derivatives; a number of TMS-carbon (TMS-C) atoms added lower than the stoichiometric one, possibly due to incomplete combustion; different TMS-C δ(13)C for di-, tri- and tetra-TMS derivatives. For soil samples, only four amino acids (leucine, valine, threonine and serine) provide reliable δ(13)C values with a total average uncertainty of 1.3 ‰. We conclude that trimethylsilyl derivatives are only suitable for determining the (13)C incorporation in amino acids within experiments using (13)C-labelled tracers but cannot be applied for amino acids with natural carbon isotope abundance until the drawbacks described here are overcome and the measured total uncertainty significantly decreased.

  16. M1 suppression in pion photoproduction on 13C

    International Nuclear Information System (INIS)

    Tiator, L.

    1983-02-01

    Recently measured anomalously low cross sections for 13 C(γ,π - ) 13 N at low energy and theta sub(π)sup(lab) = 90 degrees have been analyzed in a DWIA calculation. It has been found that the EO contribution alone is able to explain the data, so that the MI cross section is expected to vanish. Using constraints from recent magnetic electron scattering, an explanation is possible by assuming a significantly lower reduced density matrix element for spin-flip isovector transitions with angular momentum L = 2 than predicted by Cohen-Kurath

  17. Dynamically Decoupled 13C Spins in Hyperpolarized Nanodiamond

    Science.gov (United States)

    Rej, Ewa; Gaebel, Torsten; Boele, Thomas; Waddington, David; Reilly, David

    The spin-spin relaxation time, T2, which determines how long a quantum state remains coherent, is an important factor for many applications ranging from MRI to quantum computing. A common technique used in quantum information technology to extend the T2, involves averaging out certain noise spectra via dynamical decoupling sequences. Depending on the nature of the noise in the system, specific sequences, such as CPMG, UDD or KDD, can be tailored to optimize T2. Here we combine hyperpolarization techniques and dynamical decoupling sequences to extend the T2 of 13C nuclear spins in nanodiamond by three orders of magnitude.

  18. Does point-of-purchase nutrition labeling influence meal selections? A test in an Army cafeteria.

    Science.gov (United States)

    Sproul, Allen D; Canter, Deborah D; Schmidt, Jeffrey B

    2003-07-01

    This study assessed the effectiveness of nutrition labeling on sales of targeted entrees and measured the perceived influence that factors such as taste, quality, appearance, fat content, calorie content, and price had on meal selection behavior within an Army cafeteria. A quasi-experimental design was used to compare targeted entrée sales between a 1-year baseline period and two 30-day postintervention periods, after the placement of entrée nutrition labels. A brief questionnaire, distributed to 149 patrons, measured the perceived influence of the aforementioned factors on selections. Analysis of variance detected no significant differences in sales between baseline and the two intervention periods; the factors of taste and quality were rated most influential to meal selection (p < 0.000). A marketing campaign focusing on the health attributes of targeted entrée items was not successful in boosting sales. Sensory attributes (i.e., taste, quality, and appearance) appear to be more influential to meal selection.

  19. /sup 13/C-/sup 13/C spin-spin coupling constants in structural investigations. II. Conformational structure of vinyl ethers

    Energy Technology Data Exchange (ETDEWEB)

    Krivdin, L.B.; Shcherbakov, V.V.; Bzhezovskii, V.M.; Kalabin, G.A.

    1986-10-10

    The /sup 13/C-/sup 13/C spin-spin coupling constants between the carbon nuclei of the vinyl group were measured for a series of vinyl ethers. It was established that the unshared electron pairs of the oxygen atom can make a substantial stereospecific contribution to the direct /sup 13/C-/sup 13/C constants of the adjacent nuclei. The observed effect was used to establish the conformational structure of the compounds.

  20. Effects of a glucokinase activator on hepatic intermediary metabolism: study with 13C-isotopomer-based metabolomics

    OpenAIRE

    Nissim, Itzhak; Horyn, Oksana; Nissim, Ilana; Daikhin, Yevgeny; Wehrli, Suzanne L.; Yudkoff, Marc; Matschinsky, Franz M.

    2012-01-01

    GKAs (glucokinase activators) are promising agents for the therapy of Type 2 diabetes, but little is known about their effects on hepatic intermediary metabolism. We monitored the fate of 13C-labelled glucose in both a liver perfusion system and isolated hepatocytes. MS and NMR spectroscopy were deployed to measure isotopic enrichment. The results demonstrate that the stimulation of glycolysis by GKA led to numerous changes in hepatic metabolism: (i) augmented flux through the TCA (tricarboxy...

  1. Solid-state 13C NMR characterization of polyanilines

    International Nuclear Information System (INIS)

    Kaplan, S.

    1988-01-01

    13 C solid-state nuclear magnetic resonance measurements are reported for the leucoemeraldine base, emeraldine base, and emeraldine hydrochloride forms of polyaniline in order to characterize the structures of these three distinct polymers. Chemical shift assignments are facilitated by use of the cross-depolarization technique to distinguish carbons with and without directly bonded hydrogens. Comparison of the spectra of emeraldine base with those of leucoemeraldine base and air-oxidized leucoemeraldine (which partially converts to emeraldine base) establishes that emeraldine base is essentially an alternating copolymer of reduced 1A (-(C 6 H 4 )N(H)(C 6 H 4 )N(H)-) and oxidized 2A (-(C 6 H 4 )N double-bond(C 6 H 4 )double-bond N-) repeat units. The 8-12 ppm spectral line widths measured for both emeraldine base and leucoemeraldine base are attributed to local fluctuations in conformational and configurational geometries, a distribution in chain packing, and compositional defects. 13 C spin-echo measurements establish that the 60 ppM wide line from the conducting emeraldine hydrochloride is inhomogeneously broadened. It is postulated that this line width is due to local variations in charge density along the polymer backbone arising from polymer structural heterogeneity. 47 refs., 5 figs., 1 tab

  2. Near-threshold charged pion photoproduction from 13C

    International Nuclear Information System (INIS)

    LeRose, J.J.

    1981-01-01

    Differential cross sections to discrete final states have been measured for both positive and negative pion photo-production on 13 C at 90 0 in the lab at pion energies of 18, 29, and 41 MeV. Measurements were made using a fixed angle magnetic spectrometer located in the 14 0 area of the MIT Bates linear accelerator. Pions were detected using a 90 channel multi-wire proportional counter in the focal plane along with a backup array consisting of three 1/16'' thick plastic scintillator detectors and a 1/2'' thick Cerenkov detector. Positive pion photo-production cross sections were obtained for the excitation of the 3/2 - ground state and for the 3.45 MeV first excited state of 13 B. Negative pion photo-production cross sections were obtained for the excitation of the 1/2 - ground state, and the 3/2 to 3.51 MeV and 5/2 to 7.39 MeV excited states of 13 N. The measured positive pion photo-production ground state cross sections are in reasonable agreement wth theoretical calculations. However, there is a large discrepancy between the measured negative pion photoproduction ground state cross sections and the theoretical values. There are no theoretical calculations available for comparison with the excited state measurements in either positive or negative pion photoproduction on 13 C

  3. Active colloids as mobile microelectrodes for unified label-free selective cargo transport.

    Science.gov (United States)

    Boymelgreen, Alicia M; Balli, Tov; Miloh, Touvia; Yossifon, Gilad

    2018-02-22

    Utilization of active colloids to transport both biological and inorganic cargo has been widely examined in the context of applications ranging from targeted drug delivery to sample analysis. In general, carriers are customized to load one specific target via a mechanism distinct from that driving the transport. Here we unify these tasks and extend loading capabilities to include on-demand selection of multiple nano/micro-sized targets without the need for pre-labelling or surface functionalization. An externally applied electric field is singularly used to drive the active cargo carrier and transform it into a mobile floating electrode that can attract (trap) or repel specific targets from its surface by dielectrophoresis, enabling dynamic control of target selection, loading and rate of transport via the electric field parameters. In the future, dynamic selectivity could be combined with directed motion to develop building blocks for bottom-up fabrication in applications such as additive manufacturing and soft robotics.

  4. Glasshouse seedling δ13C and canopy δ13C of 8-year-old hoop pine families grown in south-east Queensland in relation to canopy δ18O, nitrogen concentration and tree growth

    International Nuclear Information System (INIS)

    Prasolova, N.V.; Saffigna, P.G.; Farquhar, G.D.

    1999-01-01

    Full text: Carbon isotope composition (δ l3 C) in C 3 plants has been theoretically and empirically linked to plant water-use efficiency (WUE). The plant δ 13 C has been suggested as an early selection criterion in plant breeding. The δ 13 C and nitrogen concentration (N mass ) of branchlet tissue for inner and outer upper canopy positions were assessed for 8-year-old hoop pine (Araucaria cunninghamii Ait.ex D.Don) trees from 23 half-sib families grown in 5 blocks of a progeny test in south-east Queensland, and for glasshouse seedlings. Tree height was positively related to N mass in branchlets of the 2 canopy positions, indicating that tree growth was limited by nitrogen deficiency. Our data indicated that water supply was also a growth-limiting factor in the hoop pine plantation of the study-area. There were considerable variations in δ 13 C and N mass between families and blocks in the field (with a heritability estimate of 0.73 for branchlet δ 13 C from the inner canopy position). Significant variation in δ 13 C was observed between positions and families of the glasshouse seedlings (with a heritability estimate of 0.66). There was also a significant difference in N mass between the 2 sampling positions in the field. The δ 13 C was positively related to N mass for the branchlets of the upper outer crown (r=0.62, p 13 C (r: 0.28-0.41, p l3 C of the 2 canopy positions for all the 115 trees sampled. There was no significant correlation between δ 13 C values for 2 seedling canopy positions at age 18 months, possibly due to restricted rooting conditions in small pots, leading to greater stomatal closure in upper canopy, which was also reflected in more positive δ 13 C. Significant correlation existed between seedling δ 13 C of upper canopy position at age 8 months and low canopy position at age 18 months. This, together with field data, demonstrated the sensitivity and reliability of δ 13 C as an indicator of plant growth environment. Strong correlation

  5. Selective labeling of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein

    Science.gov (United States)

    Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Arimura, Shin-ichi; Tsutsumi, Nobuhiro; Fukui, Kiichi; Itoh, Kazuyoshi

    2008-02-01

    We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

  6. Variations in growth, survival and carbon isotope composition (delta(13)C) among Pinus pinaster populations of different geographic origins.

    Science.gov (United States)

    Correia, Isabel; Almeida, Maria Helena; Aguiar, Alexandre; Alía, Ricardo; David, Teresa Soares; Pereira, João Santos

    2008-10-01

    To evaluate differences in growth and adaptability of maritime pine (Pinus pinaster Ait.), we studied growth, polycyclism, needle tissue carbon isotope composition (delta(13)C) as an estimate of water-use efficiency (WUE) and survival of seven populations at 10 years of age growing in a performance trial at a provenance test site in Escaroupim, Portugal. Six populations were from relatively high rainfall sites in Portugal and southwestern France (Atlantic group), and one population was from a more arid Mediterranean site in Spain. There were significant differences between some populations in total height, diameter at breast height, delta(13)C of bulk needle tissue, polycyclism and survival. A population from central Portugal (Leiria, on the Atlantic coast) was the tallest and had the lowest delta(13)C. Overall, the variation in delta(13)C was better explained by the mean minimum temperatures of the coldest month than by annual precipitation at the place of origin. Analyses of the relationships between delta(13)C and growth or survival revealed a distinct pattern for the Mediterranean population, with low delta(13)C (and WUE) associated with the lowest growth potential and reduced survival. There were significant negative correlations between delta(13)C and height or survival in the Atlantic group. Variation in polycyclism was correlated with annual precipitation at the place of origin. Some Atlantic populations maintained a high growth potential while experiencing moderate water stress. A detailed knowledge of the relationships between growth, survival and delta(13)C in contrasting environments will enhance our ability to select populations for forestry or conservation.

  7. Lack of genetic variation in tree ring delta13C suggests a uniform, stomatally-driven response to drought stress across Pinus radiata genotypes.

    Science.gov (United States)

    Rowell, Douglas M; Ades, Peter K; Tausz, Michael; Arndt, Stefan K; Adams, Mark A

    2009-02-01

    We assessed the variation in delta(13)C signatures of Pinus radiata D. Don stemwood taken from three genetic trials in southern Australia. We sought to determine the potential of using delta(13)C signatures as selection criteria for drought tolerance. Increment cores were taken from P. radiata and were used to determine the basal area increment and the delta(13)C signature of extracted cellulose. Both growth increment and cellulose delta(13)C were affected by water availability. Growth increment and delta(13)C were negatively correlated suggesting that growth was water-limited. While there was significant genetic variation in growth, there was no significant genetic variation in cellulose delta(13)C of tree rings. This suggests that different genotypes of P. radiata display significant differences in growth and yet respond similarly to drought stress. The delta(13)C response to drought stress was more due to changes in stomatal conductance than to the variation in photosynthetic capacity, and this may explain the lack of genetic variation in delta(13)C. The lack of genetic variation in cellulose delta(13)C of tree rings precludes its use as a selection criterion for drought tolerance among P. radiata genotypes.

  8. The effect of menu labeling with calories and exercise equivalents on food selection and consumption.

    Science.gov (United States)

    Platkin, Charles; Yeh, Ming-Chin; Hirsch, Kimberly; Wiewel, Ellen Weiss; Lin, Chang-Yun; Tung, Ho-Jui; Castellanos, Victoria H

    2014-01-01

    Better techniques are needed to help consumers make lower calorie food choices. This pilot study examined the effect of menu labeling with caloric information and exercise equivalents (EE) on food selection. Participants, 62 females, ages 18-34, recruited for this study, ordered a fast food meal with menus that contained the names of the food (Lunch 1 (L1), control meal). One week later (Lunch 2 (L2), experiment meal), participants ordered a meal from one of three menus with the same items as the previous week: no calorie information, calorie information only, or calorie information and EE. There were no absolute differences between groups in calories ordered from L1 to L2. However, it is noteworthy that calorie only and calorie plus exercise equivalents ordered about 16% (206 kcal) and 14% (162 kcal) fewer calories from Lunch 1 to Lunch 2, respectively; whereas, the no information group ordered only 2% (25 kcal) fewer. Menu labeling alone may be insufficient to reduce calories; however, further research is needed in finding the most effective ways of presenting the menu labels for general public.

  9. Spectrally edited 2D 13Csbnd 13C NMR spectra without diagonal ridge for characterizing 13C-enriched low-temperature carbon materials

    Science.gov (United States)

    Johnson, Robert L.; Anderson, Jason M.; Shanks, Brent H.; Fang, Xiaowen; Hong, Mei; Schmidt-Rohr, Klaus

    2013-09-01

    Two robust combinations of spectral editing techniques with 2D 13Csbnd 13C NMR have been developed for characterizing the aromatic components of 13C-enriched low-temperature carbon materials. One method (exchange with protonated and nonprotonated spectral editing, EXPANSE) selects cross peaks of protonated and nearby nonprotonated carbons, while the other technique, dipolar-dephased double-quantum/single-quantum (DQ/SQ) NMR, selects signals of bonded nonprotonated carbons. Both spectra are free of a diagonal ridge, which has many advantages: Cross peaks on the diagonal or of small intensity can be detected, and residual spinning sidebands or truncation artifacts associated with the diagonal ridge are avoided. In the DQ/SQ experiment, dipolar dephasing of the double-quantum coherence removes protonated-carbon signals; this approach also eliminates the need for high-power proton decoupling. The initial magnetization is generated with minimal fluctuation by combining direct polarization, cross polarization, and equilibration by 13C spin diffusion. The dipolar dephased DQ/SQ spectrum shows signals from all linkages between aromatic rings, including a distinctive peak from polycondensed aromatics. In EXPANSE NMR, signals of protonated carbons are selected in the first spectral dimension by short cross polarization combined with dipolar dephasing difference. This removes ambiguities of peak assignment to overlapping signals of nonprotonated and protonated aromatic carbons, e.g. near 125 ppm. Spin diffusion is enhanced by dipolar-assisted rotational resonance. Before detection, Csbnd H dipolar dephasing by gated decoupling is applied, which selects signals of nonprotonated carbons. Thus, only cross peaks due to magnetization originating from protonated C and ending on nearby nonprotonated C are retained. Combined with the chemical shifts deduced from the cross-peak position, this double spectral editing defines the bonding environment of aromatic, COO, and Cdbnd O carbons

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

    Science.gov (United States)

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

    2007-10-01

    Utilization of long-range (1)H--(15)N heteronuclear chemical shift correlation has continually grown in importance since the first applications were reported in 1995. More recently, indirect covariance NMR methods have been introduced followed by the development of unsymmetrical indirect covariance processing methods. The latter technique has been shown to allow the calculation of hyphenated 2D NMR data matrices from more readily acquired nonhyphenated 2D NMR spectra. We recently reported the use of unsymmetrical indirect covariance processing to combine (1)H--(13)C GHSQC and (1)H--(15)N GHMBC long-range spectra to yield a (13)C--(15)N HSQC-HMBC chemical shift correlation spectrum that could not be acquired in a reasonable period of time without resorting to (15)N-labeled molecules. We now report the unsymmetrical indirect covariance processing of (1)H--(13)C GHMBC and (1)H--(15)N IMPEACH spectra to afford a (13)C--(15)N HMBC-IMPEACH spectrum that has the potential to span as many as six to eight bonds. Correlations for carbon resonances long-range coupled to a protonated carbon in the (1)H--(13)C HMBC spectrum are transferred via the long-range (1)H--(15)N coupling pathway in the (1)H--(15)N IMPEACH spectrum to afford a much broader range of correlation possibilities in the (13)C--(15)N HMBC-IMPEACH correlation spectrum. The indole alkaloid vincamine is used as a model compound to illustrate the application of the method. (c) 2007 John Wiley & Sons, Ltd.

  11. Acetate and bicarbonate assimilation and metabolite formation in Chlamydomonas reinhardtii: a 13C-NMR study.

    Directory of Open Access Journals (Sweden)

    Himanshu Singh

    Full Text Available Cellular metabolite analyses by (13C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly (13C-labelled acetate ((13CH(3-COOH or CH(3-(13COOH supported that both the (13C nuclei give rise to bicarbonate and CO2(aq. The observed metabolite(s upon further incubation led to the production of starch and triacylglycerol (TAG in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2(aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2(aq, which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2(aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

  12. Reconstructing past climate using a multi-specific 13C-approach

    Science.gov (United States)

    Ferrio, Juan Pedro; Aguilera, Mónica; Voltas, Jordi

    2010-05-01

    Carbon isotope composition (δ13C) in tree-rings has become routinely used in palaeoclimatic research for the assessment of changes in water availability in seasonally dry climates. Long tree-ring chronologies, however, are relatively scarce, whereas the original climate signal of wood δ13C is usually well preserved in fossil charcoal [1, 4] Accordingly, charcoal δ13C records are an alternative to classic dendroclimatology to characterize past changes in water availability (e.g. precipitation). In this work, we explore the potential for palaeoenvironmental research of two co-occuring Mediterranean species with contrasting strategies to cope with drought [2]: Aleppo pine (Pinus halepensis Mill.) and holm oak (Quercus ilex L.). We hypothesize that the differential sensitivity of pine and oak to climate variables can be exploited to refine palaeoclimate reconstructions based on δ13C in wood or charcoal. For this purpose, we put together published tree-core-δ13C data from 40 sites across Spain [2, 3] and new δ13C data from 15 sites where both species co-existed in mixed stands. The sites were selected to represent the range of variation in thermal and precipitation regimes for these species, while avoiding any correlation between precipitation and temperature across sites. Five dominant or codominant trees were selected per site, and microcores including the most recently formed tree rings were obtained with a Trephor tool [5]. Fragments were oven-dried at 60 ° C for 48 h and milled separately to a fine powder using a ball mill (Retsch MM301, Haan, Germany) for δ13C analysis. Current meteorological data (monthly estimates of air mean temperature (minimum, mean and maximum), precipitation and solar radiation) was obtained from the Digital Climatic Atlas of the Iberian Peninsula (http://opengis.uab.es/wms/iberia/index.htm) (spatial resolution of 200 m). A family of models (either linear or exponential) best predicting monthly and annual precipitation from δ13C

  13. Foliar δ13C Showed No Altitudinal Trend in an Arid Region and Atmospheric Pressure Exerted a Negative Effect on Plant δ13C

    Directory of Open Access Journals (Sweden)

    Zixun Chen

    2017-07-01

    Full Text Available Previous studies have suggested foliar δ13C generally increases with altitude. However, some observations reported no changes or even decreased trends in foliar δ13C. We noted that all the studies in which δ13C increased with elevation were conducted in the human regions, whereas those investigations in which δ13C did not vary or decreased were conducted in areas with water stress. Thus, we proposed that the pattern of increasing δ13C with elevation is not a general one, and that δ13C may remain unchanged or decrease in plants grown in arid environments. To test the hypothesis, we sampled plants along altitude gradients on the shady and sunny slopes of Mount Tianshan characterized by arid and semiarid climates. The measurements of foliar δ13C showed no altitudinal trends for the plants grown on either of the slopes. Therefore, this study supported our hypothesis. In addition, the present study addressed the effect of atmospheric pressure on plant δ13C by accounting for the effects of temperature and precipitation on δ13C. This study found that the residual foliar δ13C increased with increasing altitude, suggesting that atmospheric pressure played a negative role in foliar δ13C.

  14. HSQC-1,n-ADEQUATE: a new approach to long-range 13C-13C correlation by covariance processing.

    Science.gov (United States)

    Martin, Gary E; Hilton, Bruce D; Willcott, M Robert; Blinov, Kirill A

    2011-10-01

    Long-range, two-dimensional heteronuclear shift correlation NMR methods play a pivotal role in the assembly of novel molecular structures. The well-established GHMBC method is a high-sensitivity mainstay technique, affording connectivity information via (n)J(CH) coupling pathways. Unfortunately, there is no simple way of determining the value of n and hence no way of differentiating two-bond from three- and occasionally four-bond correlations. Three-bond correlations, however, generally predominate. Recent work has shown that the unsymmetrical indirect covariance or generalized indirect covariance processing of multiplicity edited GHSQC and 1,1-ADEQUATE spectra provides high-sensitivity access to a (13)C-(13) C connectivity map in the form of an HSQC-1,1-ADEQUATE spectrum. Covariance processing of these data allows the 1,1-ADEQUATE connectivity information to be exploited with the inherent sensitivity of the GHSQC spectrum rather than the intrinsically lower sensitivity of the 1,1-ADEQUATE spectrum itself. Data acquisition times and/or sample size can be substantially reduced when covariance processing is to be employed. In an extension of that work, 1,n-ADEQUATE spectra can likewise be subjected to covariance processing to afford high-sensitivity access to the equivalent of (4)J(CH) GHMBC connectivity information. The method is illustrated using strychnine as a model compound. Copyright © 2011 John Wiley & Sons, Ltd.

  15. Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy

    International Nuclear Information System (INIS)

    Kohlbrenner, W.E.; Fesik, S.W.

    1985-01-01

    [99%, 1- 13 C]- and [90%, 2- 13 C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13 C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1- 13 C- or 2- 13 C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13 C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1- 13 C]- and [2- 13 C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1- 13 C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO

  16. {sup 13} C-NMR of mesquite gum

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Cristina T; Garcia, Rosangela B [Universidade Federal, Rio de Janeiro, RJ (Brazil). Inst. de Macromoleculas

    1992-12-31

    Mesquite and guar gums are galactomannans extracted from the seeds of Proposis Juliflora and Cyamopsis tetragonolobus, respectively. An experimental sample of mesquite gum and a commercial sample of guar gum were partially depolymerized by ultrasonic radiation and the produce analysed by high resolution {sup 13} C-NMR spectroscopy. The different carbon lines were resolved and their assignments were done as those reported in the literature. The galactose to mannose ratios (G/M) were estimated from the relative peak areas of the C-1 lines as G/M=61 for mesquite and G/M=0.54 for guar gum. The next nearest-neighbour probabilities (diad frequencies) of the D-galactosyl substitution to the D-mannose backbone were evaluated by integrating C-4 mannose splitted peaks. (author) 9 refs., 2 figs., 2 tabs.

  17. Exploring enhanced menu labels' influence on fast food selections and exercise-related attitudes, perceptions, and intentions.

    Science.gov (United States)

    Lee, Morgan S; Thompson, Joel Kevin

    2016-10-01

    Labeling restaurant menus with calorie counts is a popular public health intervention, but research shows these labels have small, inconsistent effects on behavior. Supplementing calorie counts with physical activity equivalents may produce stronger results, but few studies of these enhanced labels have been conducted, and the labels' potential to influence exercise-related outcomes remains unexplored. This online study evaluated the impact of no information, calories-only, and calories plus equivalent miles of walking labels on fast food item selection and exercise-related attitudes, perceptions, and intentions. Participants (N = 643) were randomly assigned to a labeling condition and completed a menu ordering task followed by measures of exercise-related outcomes. The labels had little effect on ordering behavior, with no significant differences in total calories ordered and counterintuitive increases in calories ordered in the two informational conditions in some item categories. The labels also had little impact on the exercise-related outcomes, though participants in the two informational conditions perceived exercise as less enjoyable than did participants in the no information condition, and trends following the same pattern were found for other exercise-related outcomes. The present findings concur with literature demonstrating small, inconsistent effects of current menu labeling strategies and suggest that alternatives such as traffic light systems should be explored. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Enhanced benthic activity in sandy sublittoral sediments: Evidence from 13C tracer experiments

    DEFF Research Database (Denmark)

    Bühring, Solveig I.; Ehrenhauss, Sandra; Kamp, Anja

    2006-01-01

    In situ and on-board pulse-chase experiments were carried out on a sublittoral fine sand in the German Bight (southern North Sea) to investigate the hypothesis that sandy sediments are highly active and have fast turnover rates. To test this hypothesis, we conducted a series of experiments where we...... investigated the pathway of settling particulate organic carbon through the benthic food web. The diatom Ditylum brightwellii was labelled with the stable carbon isotope 13C and injected into incubation chambers. On-board incubations lasted 12, 30 and 132 h, while the in situ experiment was incubated for 32 h....... The study revealed a stepwise short-term processing of a phytoplankton bloom settling on a sandy sediment. After the 12 h incubation, the largest fraction of recovered carbon was in the bacteria (62%), but after longer incubation times (30 and 32 h in situ) the macrofauna gained more importance (15 and 48...

  19. Carbon-11-labelling of a novel, trishomocubane-derived, high affinity and selectivity DAT ligand

    International Nuclear Information System (INIS)

    Dolle, F.; Le Helleix, St.; Peyronneau, M.A.; Saba, W.; Tournier, N.; Valette, H.; Banister, S.; Kassiou, M.

    2011-01-01

    Complete text of publication follows: Objectives: Parkinson's disease, schizophrenia, attention deficit disorder and drug abuse are related to abnormalities within the brain's dopaminergic system. The neuronal dopamine transporter (DAT) plays a key role in regulating the synaptic concentration of dopamine and thus dopamine neurotransmission in the brain. Since the DAT can be considered as a marker of the integrity and number of the presynaptic striatal dopamine-producing neurons, considerable efforts have been spent in recent years on the design and development of DAT-selective radioligands for use in Positron Emission Tomography (PET) studies. Notably, the tropane PE2I and its fluorinated analogue LBT-999 were identified as having high affinity and selectivity for the DAT over the norepinephrine transporter (NET) and the serotonin transporter (SERT). Besides tropanes, only a few bicyclic frameworks, e.g. bicyclo[2.2.2]octanes, have delivered compounds with high affinity for the DAT. Recently, novel poly-carbocyclic DAT ligands with selectivity over the NET and the SERT were reported. The lead compound of this series (1, N-methyl-N-(3-fluoro) benzyl-pentacyclo[5.4.0.0 2, 6 .0 3, 10 .0 5, 9 ] undec-8-ylamine, Ki = 1.2 nM, ≥ 8300-fold selectivity over NET and SERT) was selected as a potential candidate for imaging the DAT with PET and isotopically labelled with carbon-11 using [ 11 C]methyl triflate. Methods: The trishomocubane derivatives 1 (reference) and 2 (precursor for labelling with carbon-11) were prepared from commercially available Cookson's diketone in 6 and 7 steps, respectively. Carbon-11 labelling of 1 was performed using a TRACERLab FX-C Pro synthesizer (GEMS) and comprises (1) trapping at -10 C of [ 11 C]MeOTf in acetone (0.4 mL) containing the nor-derivative 2 (0.6-0.9 mg, free base) and aq. 3N NaOH (8 μL); (2) heating at 110 C for 2 min; (3) concentration to dryness and taking up the residue in 1.0 mL of the HPLC mobile phase; (4) purification

  20. 13C-urea breath test analyzed with infrared isotope spectrometry for the diagnosis of Helicobacter pylori

    International Nuclear Information System (INIS)

    Dahlberg, M.; Schoensby, H.

    1998-01-01

    The authors have evaluated a 13 C-urea breath test for the diagnosis of Helicobacter pylori infection. The 13 C-test was analyzed with isotope-selective non-dispersive infrared spectrometry and compared with a 14 C-urea breath test and the urease test in gastric mucosal biopsies. 46 patients were analyzed with breath tests, 23 patients were negative and 22 patients were positive with both methods. One patients was positive with 14 C-method and negative with the 13 C-urea breath test. 61 patients were analyzed with the 13 C-urea breath test and the urease test. 30 patients were negative and 30 patients were positive with both methods, whereas one patient with a negative urease test had a positive breath test. 13 C-urea breath test analyzed with isotope-selective non-dispersive infrared spectrometry is a fast, simple, non-radioactive, non-invasive, convenient and reliable method for the diagnosis of Helicobacter pylori infection. 12 refs., 2 figs., 1 tab

  1. FluxPyt: a Python-based free and open-source software for 13C-metabolic flux analyses.

    Science.gov (United States)

    Desai, Trunil S; Srivastava, Shireesh

    2018-01-01

    13 C-Metabolic flux analysis (MFA) is a powerful approach to estimate intracellular reaction rates which could be used in strain analysis and design. Processing and analysis of labeling data for calculation of fluxes and associated statistics is an essential part of MFA. However, various software currently available for data analysis employ proprietary platforms and thus limit accessibility. We developed FluxPyt, a Python-based truly open-source software package for conducting stationary 13 C-MFA data analysis. The software is based on the efficient elementary metabolite unit framework. The standard deviations in the calculated fluxes are estimated using the Monte-Carlo analysis. FluxPyt also automatically creates flux maps based on a template for visualization of the MFA results. The flux distributions calculated by FluxPyt for two separate models: a small tricarboxylic acid cycle model and a larger Corynebacterium glutamicum model, were found to be in good agreement with those calculated by a previously published software. FluxPyt was tested in Microsoft™ Windows 7 and 10, as well as in Linux Mint 18.2. The availability of a free and open 13 C-MFA software that works in various operating systems will enable more researchers to perform 13 C-MFA and to further modify and develop the package.

  2. Regioselectivity of tributyltin ether mediated alkylations. A 119Sn and 13C NMR study

    International Nuclear Information System (INIS)

    Cruzado, C.; Bernabe, M.; Martin-Lomas, M.

    1989-01-01

    The 119 Sn and 13 C NMR spectra of the stannylated species resulting from the treatment of conformationally rigid polyhydroxylated compounds with bis(tributyltin) oxide have been determined and the effect of N-methylimidazole, added as catalyst in tributyltin ether mediated regioselective alkylations, has been investigated. The observed signal intensity changes, upfield shifts, signal broadenings, and the results of variable temperature experiments have been interpreted as indicative of the selective formation of pentacoordinated tin species, involving conveniently disposed adjacent hydroxyl groups, on addition of the catalyst. On these bases, a mechanistic hypothesis for the observed regioselectivity of N-methylimidazole-catalyzed tributyltin ether mediated benzylations is proposed. 13 references, 5 tables

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

    Science.gov (United States)

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

    2008-06-01

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

  4. Using traffic light labels to improve food selection in recreation and sport facility eating environments.

    Science.gov (United States)

    Olstad, Dana Lee; Vermeer, Julianne; McCargar, Linda J; Prowse, Rachel J L; Raine, Kim D

    2015-08-01

    Many recreation and sports facilities have unhealthy food environments, however managers are reluctant to offer healthier foods because they perceive patrons will not purchase them. Preliminary evidence indicates that traffic light labeling (TLL) can increase purchase of healthy foods in away-from-home food retail settings. We examined the effectiveness of TLL of menus in promoting healthier food purchases by patrons of a recreation and sport facility concession, and among various sub-groups. TLL of all menu items was implemented for a 1-week period and sales were assessed for 1-week pre- and 1-week post-implementation of TLL (n = 2101 transactions). A subset of consumers completed a survey during the baseline (n = 322) and intervention (n = 313) periods. We assessed change in the proportion of patrons' purchases that were labeled with green, yellow and red lights from baseline to the TLL intervention, and association with demographic characteristics and other survey responses. Change in overall revenues was also assessed. There was an overall increase in sales of green (52.2% to 55.5%; p sales of red (30.4% to 27.2%; p revenues did not differ between the baseline and TLL periods. TLL of menus increased purchase of healthy, and reduced purchase of unhealthy foods in a publicly funded recreation and sport facility, with no loss of revenue. Policymakers should consider extending menu labeling laws to public buildings such as recreation and sports facilities to promote selection of healthier items. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Cohen, S.M.

    1987-01-01

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

  6. Applications of stable isotopes of /sup 2/H, /sup 13/C and /sup 15/N to clinical problems. Experience of a collaborative program at Argonne National Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Klein, P D; Szczepanik, P A; Hachey, D L [Argonne National Lab., Evanston, Ill. (USA)

    1974-08-01

    The function of the Argonne Program is to provide synthetic, analytical instrumental capability in a core facility for the clinical investigator who needs to use /sup 2/H, /sup 13/C, or /sup 15/N labelled compounds for metabolic or clinical research on pregnant women, newborn infants, young children, or for mass screening. To carry out such application development, there were six stages which were recurrent steps in every application. Five fundamental strategies should be adopted to establish the use of stable isotopes in clinical work. The instrument required for measurements was a combined gas chromatograph-mass spectrometer, and its use was schematically illustrated. Some of the successful experiences with compounds labelled by stable isotopes, such as deuterium labelled chenodeoxycholic acid, and respective /sup 13/C and /sup 15/N-labelled glycine were described. Deutrium labelled bile acid enabled easy and safe determination of the size of the bile acid pool and the replacement rate, providing clearer diagnoses for cholestatic liver disease and gallstones. /sup 13/C and /sup 15/N labelled compounds were used in clinical studies, of children with genetic disorders of amino acid metabolism, i.e., non ketotic hyperflycinemia, B/sub 12/-responsive methyl malonic acidemia, and Lesch-Nyhan syndrome. /sup 15/N-labelled glycine was also studied in a child with Lesch-Nyhan syndrome.

  7. Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons

    DEFF Research Database (Denmark)

    Brekke, Eva Marie; Walls, Anne Byriel; Schousboe, Arne

    2012-01-01

    is known about the PPP in neurons. The activity of the PPP was quantified in cultured cerebral cortical and cerebellar neurons after incubation in the presence of [2-(13)C]glucose or [3-(13)C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism...

  8. Probing slowly exchanging protein systems via {sup 13}C{sup {alpha}}-CEST: monitoring folding of the Im7 protein

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, Alexandar L.; Bouvignies, Guillaume; Kay, Lewis E., E-mail: kay@pound.med.utoronto.ca [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)

    2013-03-15

    A {sup 13}C{sup {alpha}} chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an 'observable' ground state and one or more 'invisible' excited states. Here a labeling strategy whereby [2-{sup 13}C]-glucose is the sole carbon source is exploited, producing proteins with {sup 13}C at the C{sup {alpha}} position, while the majority of residues remain unlabeled at CO or C{sup {beta}}. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state {sup 13}C{sup {alpha}} chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.

  9. The diel imprint of leaf metabolism on the δ13 C signal of soil respiration under control and drought conditions.

    Science.gov (United States)

    Barthel, Matthias; Hammerle, Albin; Sturm, Patrick; Baur, Thomas; Gentsch, Lydia; Knohl, Alexander

    2011-12-01

    Recent (13) CO(2) canopy pulse chase labeling studies revealed that photosynthesis influences the carbon isotopic composition of soil respired CO(2) (δ(13) C(SR)) even on a diel timescale. However, the driving mechanisms underlying these short-term responses remain unclear, in particular under drought conditions. The gas exchange of CO(2) isotopes of canopy and soil was monitored in drought/nondrought-stressed beech (Fagus sylvatica) saplings after (13) CO(2) canopy pulse labeling. A combined canopy/soil chamber system with gas-tight separated soil and canopy compartments was coupled to a laser spectrometer measuring mixing ratios and isotopic composition of CO(2) in air at high temporal resolution. The measured δ(13) C(SR) signal was then explained and substantiated by a mechanistic carbon allocation model. Leaf metabolism had a strong imprint on diel cycles in control plants, as a result of an alternating substrate supply switching between sugar and transient starch. By contrast, diel cycles in drought-stressed plants were determined by the relative contributions of autotrophic and heterotrophic respiration throughout the day. Drought reduced the speed of the link between photosynthesis and soil respiration by a factor of c. 2.5, depending on the photosynthetic rate. Drought slows the coupling between photosynthesis and soil respiration and alters the underlying mechanism causing diel variations of δ(13) C(SR). © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  10. Inelastic pion scattering by 13C at low energies

    International Nuclear Information System (INIS)

    Mitchell, J.H.

    1987-03-01

    Angular distributions for inelastically scattered pions were obtained for several states in 13 C at an incident energy of 65 MeV. The data include results from both π + and π - measurements. In addition, π - measurements were made at T/sub π/ = 50 MeV at one angle to give a two point fixed-q excitation function. The data are compared to theory and the data of others. As might be expected, medium corrections are shown to be considerably more important at low energies than at resonance. This is true for inelastic transitions of multipolarity 0,2 and 3. Parameters derived from an analysis of elastic pion scattering and SCX data also provide an adequate description of the inelastic transitions. The charge asymmetry in the cross sections for the 9/2 + state that was seen at resonance persists at these energies. This result is consistent with an impulse approximation treatment of the spin-flip amplitude. This is true even though the incoming energy of the pions is far below the range where the validity of an impulse treatment is expected. 65 refs., 45 figs

  11. 13C CPMAS NMR Studies of Anthocyanidins and their Glucosides

    International Nuclear Information System (INIS)

    Wolniak, M.; Wawer, I.

    2005-01-01

    Anthocyanins are responsible for red, purple or blue colours of flower petals and can be found in red or black fruits and berries. Many foods, especially red grapes and wines, aronia or blueberries contain large amounts of anthocyanins. Their health beneficial effects are related to antioxidant and radical scavenging properties. Structural analysis of anthocyanins by NMR are few, owing to the difficulty in obtaining analysable spectra for unstable, interconverting compounds, available in small amounts. Compounds studied by us were isolated from fruits and berries. 13 C CPMAS NMR spectra were recorded on a Bruker DSX-400 spectrometer for solid chlorides of: cyanidin, cyanidin 3-O-glucoside, cyanidin 3,5-O-diglucoside, pelargonidin and pelargonidin 3-O-glucoside. Dipolar dephased and short contact pulse sequences were used as an aid in the assignment of resonances in CPMAS spectra of solids. Inspection of the spectra indicates that anthocyanidins are in the form of flavylium (cationic) and not in form of the chalcone.: the resonance of C2 appears at ca. 160 ppm and C3 at ca. 135 ppm, whereas C ring opening produces C2 = O, for which chemical shift of ca. 180 ppm can be expected. A comparison of experimental (CPMAS) and predicted (GIAO DFT) shielding constants for cyanidin provided information about the orientation of OH groups, twist angle of aromatic ring B and the localization of the chloride anion.(author)

  12. Selective labelling of stromal cell-derived factor 1α with carboxyfluorescein to study receptor internalisation.

    Science.gov (United States)

    Bellmann-Sickert, Kathrin; Baumann, Lars; Beck-Sickinger, Annette G

    2010-10-01

    SDF1α plays an important role in the regeneration of injured tissue after ischemia or stroke by inducing the migration of progenitor cells. In order to study the function of this therapeutically relevant chemokine site-specific protein labelling is of great interest. However, modification of SDF1α is complicated because of its complex tertiary structure. Here, we describe the first site-specific fluorescent modification of SDF1α by EPL. We recombinantly expressed SDF1α (1-49) by intein-mediated protein expression. The C-terminal peptide SDF1α (50-68) was synthesised by SPPS and selectively labelled with carboxyfluorescein at Lys(56). In a cell migration assay, M-[K(56)(CF)]SDF1α showed a clear potency to induce chemotaxis of human T-cell leukaemia cells. Microscopic analysis on HEK293 cells transfected with the CXCR4 revealed specific binding of the fluorescent ligand. Furthermore, receptor-induced internalisation of the ligand could be visualised. These results show that site-specific modification of SDF1α yields in a biologically functional molecule that allows the characterisation of CXCR4 production of cells on a molecular level. © 2010 European Peptide Society and John Wiley & Sons, Ltd.

  13. Analysis of 13C-mixed triacylglycerol in stool by bulk (EA-IRMS) and compound specific (GC/MS) methods.

    Science.gov (United States)

    Slater, C; Ling, S C; Preston, T; Weaver, L T

    2002-06-01

    This paper was presented in poster form at the 17th International Congress of Nutrition, August 27-31, Vienna, Austria (Annals of Nutrition & Metabolism 2001; 45(Suppl.1):349). Some of the data were also presented in poster form at the British Society of Gastroenterology Meeting, March 18-21, Glasgow, UK (Gut 2001; 48(Suppl.1):A91). The 13C-mixed triacylglycerol (MTG) breath test is used to measure intraluminal fat digestion. In normal digestion, 20-40% of the ingested 13C label is recovered in breath CO2. We aimed to identify the proportions of ingested label excreted in stool, as well as breath following ingestion of 13C-MTG by children with impaired exocrine pancreatic function and healthy controls. 13C enrichment of breath samples was measured by continuous flow isotope ratio mass spectrometry (IRMS) and cumulative percent dose recovered (cPDR) in 10 h was calculated. Total 13C of a faecal fat extract from each stool was measured by elemental analyser-IRMS, and 13C enrichment and concentration of the TBDMS derivative of octanoic acid was measured by GC/MS after hydrolysis of the fat extract. Stool 5-day cPDR was calculated. Mean breath cPDR was 35%. Mean cPDR in stool by combustion-IRMS and GC/ MS, respectively, was 0.8% and 1.0%. Therefore, the remaining 64% of the 13C label must remain in the body and variability in breath cPDR is due to postabsorptive rather than predigestive factors.

  14. Preliminary study on the relationship between trends of tree-ring δ 13C series and site conditions

    International Nuclear Information System (INIS)

    Chen Tuo; Qin Dahe; Liu Xiaohong; Ren Jiawen

    2001-01-01

    The long-term trends of tree-ring δ 13 C series, taken respectively from Qilian of Qinghai and Zhaosu and Aleitai of xinjiang were compared. The results showed that a similar trend existed between Qilian series and Zhaosu series, while there was a significant difference between them and Aleitai series. The authors' analysis indicated that the site difference of the trends of tree-ring δ 13 C series was mainly associated with 'canopy effects' of tree growth. It is suggested that tree samples should be selected from sparse forests or the sampled tree foliage high above the whole canopy when the history of δ 13 C of atmospheric CO 2 was reconstructed by tree-ring series

  15. Positional enrichment by proton analysis (PEPA). A one-dimensional "1H-NMR approach for "1"3C stable isotope tracer studies in metabolomics

    International Nuclear Information System (INIS)

    Vinaixa, Maria; Yanes, Oscar; Rodriguez, Miguel A.; Capellades, Jordi; Aivio, Suvi; Stracker, Travis H.; Gomez, Josep; Canyellas, Nicolau

    2017-01-01

    A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of "1"3C-satellite peaks using 1D-"1H-NMR spectra. In comparison with "1"3C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of "1"3C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of "1H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts. (copyright 2017 The Authors. Published by Wiley-VCH Verlag GmbH and Co. KGaA.)

  16. Positional enrichment by proton analysis (PEPA). A one-dimensional {sup 1}H-NMR approach for {sup 13}C stable isotope tracer studies in metabolomics

    Energy Technology Data Exchange (ETDEWEB)

    Vinaixa, Maria; Yanes, Oscar [Department of Electronic Engineering-Universitat Rovira i Virgili, Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Reus (Spain); Rodriguez, Miguel A.; Capellades, Jordi [Universitat Rovira i Virgili, Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM), Reus (Spain); Aivio, Suvi; Stracker, Travis H. [Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (Spain); Gomez, Josep; Canyellas, Nicolau [Department of Electronic Engineering-, Universitat Rovira i Virgili, Tarragona (Spain)

    2017-03-20

    A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of {sup 13}C-satellite peaks using 1D-{sup 1}H-NMR spectra. In comparison with {sup 13}C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of {sup 13}C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of {sup 1}H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts. (copyright 2017 The Authors. Published by Wiley-VCH Verlag GmbH and Co. KGaA.)

  17. Selective cell-surface labeling of the molecular motor protein prestin

    International Nuclear Information System (INIS)

    McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

    2011-01-01

    Highlights: → Trafficking to the plasma membrane is required for prestin function. → Biotin acceptor peptide (BAP) was fused to prestin through a transmembrane domain. → BAP-prestin can be metabolically labeled with biotin in HEK293 cells. → Biotin-BAP-prestin allows for selective imaging of fully trafficked prestin. → The biotin-BAP-prestin displays voltage-sensitive activity. -- Abstract: Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

  18. 1H, 13C and 15N chemical shift assignments of the thioredoxin from the obligate anaerobe Desulfovibrio vulgaris Hildenborough.

    Science.gov (United States)

    Garcin, Edwige B; Bornet, Olivier; Pieulle, Laetitia; Guerlesquin, Françoise; Sebban-Kreuzer, Corinne

    2011-10-01

    Thioredoxins are ubiquitous key antioxidant enzymes which play an essential role in cell defense against oxidative stress. They maintain the redox homeostasis owing to the regulation of thiol-disulfide exchange. In the present paper, we report the full resonance assignments of (1)H, (13)C and (15)N atoms for the reduced and oxidized forms of Desulfovibrio vulgaris Hildenborough thioredoxin 1 (Trx1). 2D and 3D heteronuclear NMR experiments were performed using uniformly (15)N-, (13)C-labelled Trx1. Chemical shifts of 97% of the backbone and 90% of the side chain atoms were obtained for the oxidized and reduced form (BMRB deposits with accession number 17299 and 17300, respectively).

  19. Structural properties of carbon nanotubes derived from 13C NMR

    KAUST Repository

    Abou-Hamad, E.

    2011-10-10

    We present a detailed experimental and theoretical study on how structural properties of carbon nanotubes can be derived from 13C NMR investigations. Magic angle spinning solid state NMR experiments have been performed on single- and multiwalled carbon nanotubes with diameters in the range from 0.7 to 100 nm and with number of walls from 1 to 90. We provide models on how diameter and the number of nanotube walls influence NMR linewidth and line position. Both models are supported by theoretical calculations. Increasing the diameter D, from the smallest investigated nanotube, which in our study corresponds to the inner nanotube of a double-walled tube to the largest studied diameter, corresponding to large multiwalled nanotubes, leads to a 23.5 ppm diamagnetic shift of the isotropic NMR line position δ. We show that the isotropic line follows the relation δ = 18.3/D + 102.5 ppm, where D is the diameter of the tube and NMR line position δ is relative to tetramethylsilane. The relation asymptotically tends to approach the line position expected in graphene. A characteristic broadening of the line shape is observed with the increasing number of walls. This feature can be rationalized by an isotropic shift distribution originating from different diamagnetic shielding of the encapsulated nanotubes together with a heterogeneity of the samples. Based on our results, NMR is shown to be a nondestructive spectroscopic method that can be used as a complementary method to, for example, transmission electron microscopy to obtain structural information for carbon nanotubes, especially bulk samples.

  20. The effect of feeding on CO2 production and energy expenditure in ponies measured by indirect calorimetry and the 13C-bicarbonate technique.

    Science.gov (United States)

    Jensen, R B; Kyrstein, T D; Junghans, P; Tauson, A H

    2015-11-01

    Energy expenditure (EE) can be estimated based on respiratory gas exchange measurements, traditionally done in respiration chambers by indirect calorimetry (IC). However, the (13)C-bicarbonate technique ((13)C-BT) might be an alternative minimal invasive method for estimation of CO(2) production and EE in the field. In this study, four Shetland ponies were used to explore the effect of feeding on CO(2) production and EE measured simultaneously by IC and (13)C-BT. The ponies were individually housed in respiration chambers and received either a single oral or intravenous (IV) bolus dose of (13)C-labelled sodium bicarbonate (NaH(13)CO(3)). The ponies were fed haylage 3 h before (T(-3)), simultaneously with (T(0)) or 3 h after (T(+3)) administration of (13)C-bicarbonate. The CO(2) produced and O(2) consumed by the ponies were measured for 6 h with both administration routes of (13)C-bicarbonate at the three different feeding times. Feeding time affected the CO(2) production (P<0.001) and O(2) consumption (P<0.001), but not the respiratory quotient (RQ) measured by IC. The recovery factor (RF) of (13)C in breath CO(2) was affected by feeding time (P<0.01) and three different RF were used in the calculation of CO(2) production measured by 13C-BT. An average RQ was used for the calculations of EE. There was no difference between IC and (13)C-BT for estimation of CO(2) production. An effect of feeding time (P<0.001) on the estimated EE was found, with higher EE when feed was offered (T(0) and T(+3)) compared with when no feed was available (T -3) during measurements. In conclusion, this study showed that feeding time affects the RF and measurements of CO(2) production and EE. This should be considered when the (13)C-BT is used in the field. IV administration of (13)C-bicarbonate is recommended in future studies with horses to avoid complex (13)C enrichment-time curves with maxima and shoulders as observed in several experiments with oral administration of (13)C-bicarbonate.

  1. Conformational analysis of capsaicin using 13C, 15N MAS NMR, GIAO DFT and GA calculations

    Science.gov (United States)

    Siudem, Paweł; Paradowska, Katarzyna; Bukowicki, Jarosław

    2017-10-01

    Capsaicin produced by plants from genus Capsicum exerts multiple pharmacological effects and has found applications in food and pharmaceutical industry. The alkaloid was studied by a combined approach: solid-state NMR, GA conformational search and GIAO DFT methods. The 13C CPMAS NMR spectra were recorded using variable contact time and dipolar dephasing experiments. The results of cross-polarization (CP) kinetics, such as TCP values and long T1ρH (100-200 ms), indicated that the capsaicin molecule is fairly mobile, especially at the end of the aliphatic chain. The15N MAS NMR spectrum showed one narrow signal at -255 ppm. Genetic algorithm (GA) search with multi modal optimization was used to find low-energy conformations of capsaicin. Theoretical GIAO DFT calculations were performed using different basis sets to characterize five selected conformations. 13C CPMAS NMR was used as a validation method and the experimental chemical shifts were compared with those calculated for selected stable conformers. Conformational analysis suggests that the side chain can be bent or extended. A comparison of the experimental and the calculated chemical shifts indicates that solid capsaicin does not have the same structure as those established by PWXRD.

  2. Uptake of 13C-glucose by cell suspensions of carrot (Daucus carota) measured by in vivo NMR: Cycling of triose, pentose- and hexose-phosphates

    NARCIS (Netherlands)

    Krook, J.; Vreugdenhil, D.; Dijkema, C.; Plas, van der L.H.W.

    2000-01-01

    After a lag phase of 2 days, batch-grown cells of carrot (Daucus carota L.) cv. Flakkese entered the exponential growth phase and started to accumulate sucrose and hexoses. Short-term feeding 13C-glucose in this period resulted in only minor labelling of sucrose or fructose. CO2 production from

  3. Interaction of storage carbohydrates and other cyclic fluxes with central metabolism : A quantitative approach by non-stationary 13C metabolic flux analysis

    NARCIS (Netherlands)

    Suarez-Mendez, C. A.; Hanemaaijer, M.; ten Pierick, Angela; Wolters, J. C.; Heijnen, J.J.; Wahl, S. A.

    2016-01-01

    13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307 h-1) are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids), which are

  4. Analysis of [U-13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques

    NARCIS (Netherlands)

    Schierbeek, H.; Moerdijk-Poortvliet, T.C.W.; van den Akker, C.H.P.; te Braake, F.W.J.; Boschker, H.T.S.; van Goudoever, J.B.

    2009-01-01

    The use of stable isotope labelled glucose provides insight into glucose metabolism. The 13C-isotopic enrichment of glucose is usually measured by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). However, in both techniques

  5. The synthesis of [2-13C]2-nitropropane at room temperature and at atmospheric pressure

    OpenAIRE

    Jacquemijns M; Zomer G

    1990-01-01

    In this report the synthesis of [2-13C]2-nitropropane at room temperature is described. [2-13C]Acetone was converted into the oxime with hydroxy hydrochloridelamine and sodium carbonate. Treatment with hypobromic acid resulted in 2-13C]2-bromo-2-nitropropane. Hydrogenation with sodium borohydride gave [2-13C]2-nitropropane in 14,3% overall yield.

  6. Synthesis of deleobuvir, a potent hepatitis C virus polymerase inhibitor, and its major metabolites labeled with carbon-13 and carbon-14.

    Science.gov (United States)

    Latli, Bachir; Hrapchak, Matt; Chevliakov, Maxim; Li, Guisheng; Campbell, Scot; Busacca, Carl A; Senanayake, Chris H

    2015-05-30

    Deleobuvir, (2E)-3-(2-{1-[2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]cyclobutyl}-1-methyl-1H-benzimidazol-6-yl)prop-2-enoic acid (1), is a non-nucleoside, potent, and selective inhibitor of hepatitis C virus NS5B polymerase. Herein, we describe the detailed synthesis of this compound labeled with carbon-13 and carbon-14. The synthesis of its three major metabolites, namely, the reduced double bond metabolite (2) and the acyl glucuronide derivatives of (1) and (2), is also reported. Aniline-(13) C6 was the starting material to prepare butyl (E)-3-(3-methylamino-4-nitrophenyl-(13) C6 )acrylate [(13) C6 ]-(11) in six steps. This intermediate was then used to obtain [(13) C6 ]-(1) and [(13) C6 ]-(2) in five and four more steps, respectively. For the radioactive synthesis, potassium cyanide-(14) C was used to prepare 1-cylobutylaminoacid [(14) C]-(23) via Buchrer-Bergs reaction. The carbonyl chloride of this acid was then used to access both [(14) C]-(1) and [(14) C]-(2) in four steps. The acyl glucuronide derivatives [(13) C6 ]-(3), [(13) C6 ]-(4) and [(14) C]-(3) were synthesized in three steps from the acids [(13) C6 ]-(1), [(13) C6 ]-(2) and [(14) C]-(1) using known procedures. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Synthesis of isotopically labelled salicylates

    International Nuclear Information System (INIS)

    Hawkins, D.R.; Pryor, R.W.

    1981-01-01

    [ 13 C-carboxyl]Salicylic acid has been prepared by carbonation of 2-benzyloxybromobenzene followed by reductive debenzylation. Deuterium and tritium labelled salicylic acid and 2 H 2 / 13 C-salicylic acid were prepared by reduction of the 3,5-dibromo derivatives using Raney Ni-Al. Deuterium labelled salicylic acid containing up to four deuterium atoms was prepared by catalytic exchange with Raney Ni-Al in 5% NaOD/D 2 O. (author)

  8. Espiritu Santo, Vanuatu Stable Isotope (delta 18O, delta 13C) Data for 1806 to 1979

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Site: Espiritu Santo Island, Vanuatu, 15S, 167E. 173 year record of d18O and d13C. Variable names: QSR Age, QSR 13C, QSR 18O, GRL Age, GRL Qtrly 13C, GRL Qtrly 18O,...

  9. 40 CFR 721.6505 - Polymers of C13C15 oxoalcohol ethoxolates.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Polymers of C13C15 oxoalcohol... Specific Chemical Substances § 721.6505 Polymers of C13C15 oxoalcohol ethoxolates. (a) Chemical substance... polymers of C13C15 oxoalcohol ethoxolates (PMNs P-96-950/951) are subject to reporting under this section...

  10. The synthesis of [2-13C]2-nitropropane at room temperature and at atmospheric pressure

    NARCIS (Netherlands)

    Jacquemijns M; Zomer G

    1990-01-01

    In this report the synthesis of [2-13C]2-nitropropane at room temperature is described. [2-13C]Acetone was converted into the oxime with hydroxy hydrochloridelamine and sodium carbonate. Treatment with hypobromic acid resulted in 2-13C]2-bromo-2-nitropropane. Hydrogenation with sodium borohydride

  11. Synthesis and Physicochemical Properties of [19,20-13C]-17α-Ethinylestradiol

    NARCIS (Netherlands)

    Kraan, G.P.B.; Drayer, N.M.; Kruizinga, W.H.; Vaalburg, W.; Hummelen, J.C.

    1989-01-01

    13C2-17α-ethinylestradiol (13C2-EE2) was synthesized from estrone and 13C2-C2H2-gas to measure the metabolic clearance rate and the plasma concentration of 17α-ethinylestradiol (EE2) in tall girls, who are treated with high dosages of this estrogen. Interesting characteristics determined by (i) MS:

  12. Label free selective detection of estriol using graphene oxide-based fluorescence sensor

    Science.gov (United States)

    Kushwaha, H. S.; Sao, Reshma; Vaish, Rahul

    2014-07-01

    Water-soluble and fluorescent Graphene oxide (GO) is biocompatible, easy, and economical to synthesize. Interestingly, GO is also capable of quenching fluorescence. On the basis of its fluorescence and quenching abilities, GO has been reported to serve as an energy acceptor in a fluorescence resonance energy transfer (FRET) sensor. GO-based FRET biosensors have been widely reported for sensing of proteins, nucleic acid, ATP (Adenosine triphosphate), etc. GO complexes with fluorescent dyes and enzymes have been used to sense metal ions. Graphene derivatives have been used for sensing endocrine-disrupting chemicals like bisphenols and chlorophenols with high sensitivity and good reproducibility. On this basis, a novel GO based fluorescent sensor has been successfully designed to detect estriol with remarkable selectivity and sensitivity. Estriol is one of the three estrogens in women and is considered to be medically important. Estriol content of maternal urine or plasma acts as an important screening marker for estimating foetal growth and development. In addition, estriol is also used as diagnostic marker for diseases like breast cancer, osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, etc. In this present study, we report for the first time a rapid, sensitive with detection limit of 1.3 nM, selective and highly biocompatible method for label free detection of estriol under physiological conditions using fluorescence assay.

  13. Espectroscopia de Ressonância Magnética Nuclear de 13C no estudo de rotas biossintéticas de produtos naturais 13C Nuclear Magnetic Resonance spectroscopy in the studies of biosythetic routes of natural products

    Directory of Open Access Journals (Sweden)

    Fernando César de Macedo Júnior

    2007-02-01

    Full Text Available During the last five decades, as a result of an interaction between natural product chemistry, synthetic organic chemistry, molecular biology and spectroscopy, scientists reached an extraordinary level of comprehension about the natural processes by which living organisms build up complex molecules. In this context, 13C nuclear magnetic resonance spectroscopy, allied with isotopic labeling, played a determinant role. Nowadays, the widespread use of modern NMR techniques allows an even more detailed picture of the biochemical steps by accurate manipulation of the atomic nuclei. This article focuses on the development of such techniques and their impact on biosynthetic studies.

  14. Determination of fructose metabolic pathways in normal and fructose-intolerant children: A 13C NMR study using [U-13C]fructose

    International Nuclear Information System (INIS)

    Gopher, A.; Lapidot, A.; Vaisman, N.; Mandel, H.

    1990-01-01

    An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U- 13 C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13 C NMR spectra of plasma glucose. Significantly lower values (∼3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13 C NMR measurement of plasma [ 13 C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13 C atoms at glucose C-4 ( 13 C 3 - 13 C 4 - 13 C 5 ) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only ∼50% of the total amount of hepatic fructose conversion to glucose. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [ 13 C]glucose formation from a trace amount of [U- 13 C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism

  15. 13C/14C dual isotope breath test measurement of gastric emptying in normal subjects and patients

    International Nuclear Information System (INIS)

    Chew, G.; Bartholomeusz, F.D.L.; Bellon, M.; Chatterton, B.E.

    2000-01-01

    Full text: A more flexible alternative to dual isotope scintigraphy for gastric emptying involves measuring breath C0 2 after administration of absorbable tracers. Method: six patients were given 100g hamburger labelled with 25 MBq 99 Tc m sulphur colloid and 74 KBq 14 C octanoic acid, and 150 ml 10% glucose drink labelled with 8 MBq 67 Ga citrate and ISO mg 13 C acetate and seven normals with 14 C and 13 C labels only. Breath was collected at baseline and then regularly for four hours. The 14 CO 2 and 13 CO 2 activity was measured with liquid scintillation counting and mass spectroscopy. The times to maximum 14 CO 2 , and 13 CO 2 , T max, were determined. Results: Comparison was made between 14 CO 2 T max with scintigraphic retention of 99 Tc m at 100 minutes (SR100m) and 13 CO 2 T max with the scintigraphic half-clearance time of 67 Ga (scint T1/2). In conclusion 14 CO 2 T max and 13 CO 2 T max correlate significantly with SR100m and scint T1/2 respectively. The normal threshold is between 165 and 180 minutes for 14 Co 2 T max; probably > 40 minutes for 13 CO 2 T max but overlap exists between normal and abnormal results in this small preliminary study. Recruitment is to continue to better define normal ranges. Copyright (2000) The Australian and New Zealand Society of Nuclear Medicine Inc

  16. Acetylenes bearing Aromatic Terminal Groups. : II 13C-NMR Spectra of Monosubstituted Diphenylacetylenes

    OpenAIRE

    野本, 健雄; Nomoto, Takeo

    1986-01-01

    Six monosubstituted diphenylacetylenes, p-X-C6H4-C≡C-C6H5 1 (Ⅹ=NMe2, NH2, OMe, Cl, and NO2), were synthesized, and 13C-NMR spectra of their acetylenic carbons were measured. Hammett plots of the chemical shifts of the acetylenic α-13C and β-13C (against substituent constants σ) respectively showed a linear relationship, eXCept for β-13C on NMe2 and NH2 groups. The effects of substituents on 13C-Chemical shifts of diphenylacetylenes and effeciency of the C≡C bonds in transmitting the substitue...

  17. 13C, 1H spin-spin coupling constants. Pt. 4

    International Nuclear Information System (INIS)

    Aydin, R.; Guenther, H.

    1979-01-01

    One-bond, geminal, and vicinal 13 C, 1 H coupling constants have been determined for adamantane using α-and β-[D]adamantane and the relation sup(n)J( 13 C, 1 H)=6,5144sup(n)J( 13 C, 2 H) for the conversion of the measured sup(n)J( 13 C, 2 H) values. It is shown that the magnitude of 3 Jsub(trans) is strongly influenced by the substitution pattern. Relative H,D isotope effects for 13 C chemical shifts are given. (orig.) [de

  18. High-level 13C-enrichment of random and synchronous populations of Chlamydomonas reinhardii

    International Nuclear Information System (INIS)

    Price, R.L.; Crissman, H.A.; Martin, J.C.; Kollman, V.H.

    1975-01-01

    The alga Chlamydomonas reinhardii was grown in suspension culture at high levels of 13 C-enrichment (98 mol percent) both in synchronous and random populations for the purpose of investigating possible macro- and ultrastructural changes in the cell as induced by essentially total carbon replacement. The algae, grown in spinner flasks, were analyzed using a newly developed multiparameter flow-system technique applied to characterizing various algal genera. The versatility of this technique provides for measuring and processing several cell characteristics simultaneously and separating cells according to selected combinations of parameters. In these studies, cell volume (by Coulter aperture) and DNA and chlorophyll content were determined simultaneously. Cell ultrastructure was examined at various levels of isotope enrichment and time periods by electron microscopy. The data presented for synchronous growth of this organism demonstrate the absence of biological effects (considering the parameters measured) due to the almost total replacement of cellular 12 C with 13 C. Interpretational problems encountered when looking for biological effects on random populations are discussed

  19. Preparation of [13C3]-melamine and [13C3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Varelis, P; Jeskelis, R

    2008-10-01

    For the determination of melamine and cyanuric acid the labelled internal standards [(13)C(3)]-melamine and [(13)C(3)]-cyanuric acid were synthesized using the common substrate [(13)C(3)]-cyanuric chloride by reaction with ammonia and acidified water, respectively. Standards with excellent isotopic and chemical purities were obtained in acceptable yields. These compounds were used to develop an isotope dilution liquid chromatography/mass spectrometry (LC/MS) method to determine melamine and cyanuric acid in catfish, pork, chicken, and pet food. The method involved extraction into aqueous methanol, liquid-liquid extraction and ion exchange solid phase clean-up, with normal phase high-performance liquid chromatography (HPLC) in the so-called hydrophilic interaction mode. The method had a limit of detection (LOD) of 10 microg kg(-1) for both melamine and cyanuric acid in the four foods with a percentage coefficient of variation (CV) of less than 10%. The recovery of the method at this level was in the range of 87-110% and 96-110% for melamine and cyanuric acid, respectively.

  20. (13)C MRS of human brain at 7 Tesla using [2-(13)C]glucose infusion and low power broadband stochastic proton decoupling.

    Science.gov (United States)

    Li, Shizhe; An, Li; Yu, Shao; Ferraris Araneta, Maria; Johnson, Christopher S; Wang, Shumin; Shen, Jun

    2016-03-01

    Carbon-13 ((13)C) MR spectroscopy (MRS) of the human brain at 7 Tesla (T) may pose patient safety issues due to high radiofrequency (RF) power deposition for proton decoupling. The purpose of present work is to study the feasibility of in vivo (13)C MRS of human brain at 7 T using broadband low RF power proton decoupling. Carboxylic/amide (13)C MRS of human brain by broadband stochastic proton decoupling was demonstrated on a 7 T scanner. RF safety was evaluated using the finite-difference time-domain method. (13)C signal enhancement by nuclear Overhauser effect (NOE) and proton decoupling was evaluated in both phantoms and in vivo. At 7 T, the peak amplitude of carboxylic/amide (13)C signals was increased by a factor of greater than 4 due to the combined effects of NOE and proton decoupling. The 7 T (13)C MRS technique used decoupling power and average transmit power of less than 35 watts (W) and 3.6 W, respectively. In vivo (13)C MRS studies of human brain can be performed at 7 T, well below the RF safety threshold, by detecting carboxylic/amide carbons with broadband stochastic proton decoupling. © 2015 Wiley Periodicals, Inc.

  1. Hydrogen atom scrambling in selectively labeled anionic peptides upon collisional activation by MALDI tandem time-of-flight mass spectrometry

    DEFF Research Database (Denmark)

    Bache, Nicolai; Rand, Kasper Dyrberg; Roepstorff, Peter

    2008-01-01

    have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens ((1)H....../(2)H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution....

  2. Site-specific 13C content by quantitative isotopic 13C Nuclear Magnetic Resonance spectrometry: A pilot inter-laboratory study

    International Nuclear Information System (INIS)

    Chaintreau, Alain; Fieber, Wolfgang; Sommer, Horst; Gilbert, Alexis; Yamada, Keita; Yoshida, Naohiro; Pagelot, Alain; Moskau, Detlef; Moreno, Aitor; Schleucher, Jürgen; Reniero, Fabiano; Holland, Margaret; Guillou, Claude; Silvestre, Virginie; Akoka, Serge; Remaud, Gérald S.

    2013-01-01

    Graphical abstract: -- Highlights: •First ring test on isotopic 13 C NMR spectrometry. •Evaluation of the intra- and inter-variability of the NMR spectrometers used. •Definition of a protocol for qualification of the performance of the spectrometer. -- Abstract: Isotopic 13 C NMR spectrometry, which is able to measure intra-molecular 13 C composition, is of emerging demand because of the new information provided by the 13 C site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic 13 C NMR as a routine tool. This paper describes the first collaborative study of intra-molecular 13 C composition by NMR. The main goals of the ring test were to establish intra- and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic 13 C NMR was then assessed on vanillin from three different origins associated with specific δ 13 C i profiles. The standard deviation was, on average, between 0.9 and 1.2‰ for intra-variability. The highest standard deviation for inter-variability was 2.1‰. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of δ 13 C i in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results

  3. Site-specific {sup 13}C content by quantitative isotopic {sup 13}C Nuclear Magnetic Resonance spectrometry: A pilot inter-laboratory study

    Energy Technology Data Exchange (ETDEWEB)

    Chaintreau, Alain; Fieber, Wolfgang; Sommer, Horst [Firmenich SA, Corporate R and D Division, P.O. Box 239, 1211 Geneva 8 (Switzerland); Gilbert, Alexis; Yamada, Keita [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Yoshida, Naohiro [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Earth-Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8551 (Japan); Pagelot, Alain [Bruker Biospin SAS, 34 rue de l‘Industrie, 67166 Wissembourg Cedex (France); Moskau, Detlef; Moreno, Aitor [Bruker Biospin AG, Industriestrasse 26, 8117 Fällanden (Switzerland); Schleucher, Jürgen [Department of Medical Biochemistry and Biophysics, Umeå University, S-90187 Umeå (Sweden); Reniero, Fabiano; Holland, Margaret; Guillou, Claude [European Commission, Joint Research Centre – Institute for Health and Consumer Protection, via E. Fermi 2749, I-21027 Ispra (Italy); Silvestre, Virginie; Akoka, Serge [EBSI team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 rue de la Houssinière, BP 92208, F-44322 Nantes cedex 3 (France); Remaud, Gérald S., E-mail: gerald.remaud@univ-nantes.fr [EBSI team, Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), University of Nantes-CNRS UMR 6230, 2 rue de la Houssinière, BP 92208, F-44322 Nantes cedex 3 (France)

    2013-07-25

    Graphical abstract: -- Highlights: •First ring test on isotopic {sup 13}C NMR spectrometry. •Evaluation of the intra- and inter-variability of the NMR spectrometers used. •Definition of a protocol for qualification of the performance of the spectrometer. -- Abstract: Isotopic {sup 13}C NMR spectrometry, which is able to measure intra-molecular {sup 13}C composition, is of emerging demand because of the new information provided by the {sup 13}C site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic {sup 13}C NMR as a routine tool. This paper describes the first collaborative study of intra-molecular {sup 13}C composition by NMR. The main goals of the ring test were to establish intra- and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic {sup 13}C NMR was then assessed on vanillin from three different origins associated with specific δ{sup 13}C{sub i} profiles. The standard deviation was, on average, between 0.9 and 1.2‰ for intra-variability. The highest standard deviation for inter-variability was 2.1‰. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of δ{sup 13}C{sub i} in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.

  4. Chemical Composition and Labeling of Substances Marketed as Selective Androgen Receptor Modulators and Sold via the Internet.

    Science.gov (United States)

    Van Wagoner, Ryan M; Eichner, Amy; Bhasin, Shalender; Deuster, Patricia A; Eichner, Daniel

    2017-11-28

    Recent reports have described the increasing use of nonsteroidal selective androgen receptor modulators, which have not been approved by the US Food and Drug Administration (FDA), to enhance appearance and performance. The composition and purity of such products is not known. To determine the chemical identity and the amounts of ingredients in dietary supplements and products marketed and sold through the internet as selective androgen receptor modulators and compare the analyzed contents with product labels. Web-based searches were performed from February 18, 2016, to March 25, 2016, using the Google search engine on the Chrome and Internet Explorer web browsers to identify suppliers selling selective androgen receptor modulators. The products were purchased and the identities of the compounds and their amounts were determined from April to August 2016 using chain-of-custody and World Anti-Doping Association-approved analytical procedures. Analytical findings were compared against the label information. Products marketed and sold as selective androgen receptor modulators. Chemical identities and the amount of ingredients in each product marketed and sold as selective androgen receptor modulators. Among 44 products marketed and sold as selective androgen receptor modulators, only 23 (52%) contained 1 or more selective androgen receptor modulators (Ostarine, LGD-4033, or Andarine). An additional 17 products (39%) contained another unapproved drug, including the growth hormone secretagogue ibutamoren, the peroxisome proliferator-activated receptor-δ agonist GW501516, and the Rev-ErbA agonist SR9009. Of the 44 tested products, no active compound was detected in 4 (9%) and substances not listed on the label were contained in 11 (25%). In only 18 of the 44 products (41%), the amount of active compound in the product matched that listed on the label. The amount of the compounds listed on the label differed substantially from that found by analysis in 26 of 44 products

  5. Quantifying the Contribution of Grape Hexoses to Wine Volatiles by High-Precision [U13C]-Glucose Tracer Studies

    Science.gov (United States)

    Nisbet, Mark A.; Tobias, Herbert J.; Brenna, J. Thomas; Sacks, Gavin L.; Mansfield, Anna Katharine

    2016-01-01

    Many fermentation volatiles important to wine aroma potentially arise from yeast metabolism of hexose sugars, but assessing the relative importance of these pathways is challenging due to high endogenous hexose substrate concentrations. To overcome this problem, gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) was used to measure high-precision 13C/12C isotope ratios of volatiles in wines produced from juices spiked with tracer levels (0.01–1 APE) of uniformly labeled [U-13C]-glucose. The contribution of hexose to individual volatiles was determined from the degree of 13C enrichment. As expected, straight-chain fatty acids and their corresponding ethyl esters were derived almost exclusively from hexoses. Most fusel alcohols and their acetate esters were also majority hexose-derived, indicating the importance of anabolic pathways for their formation. Only two compounds were not derived primarily from hexoses (hexanol and isobutyric acid). This approach can be extended to other food systems or substrates for studying precursor–product relationships. PMID:24960193

  6. A voluntary nutrition labeling program in restaurants: Consumer awareness, use of nutrition information, and food selection.

    Science.gov (United States)

    White, Christine M; Lillico, Heather G; Vanderlee, Lana; Hammond, David

    2016-12-01

    Health Check (HC) was a voluntary nutrition labeling program developed by the Heart and Stroke Foundation of Canada as a guide to help consumers choose healthy foods. Items meeting nutrient criteria were identified with a HC symbol. This study examined the impact of the program on differences in consumer awareness and use of nutritional information in restaurants. Exit surveys were conducted with 1126 patrons outside four HC and four comparison restaurants in Ontario, Canada (2013). Surveys assessed participant noticing of nutrition information, influence of nutrition information on menu selection, and nutrient intake. Significantly more patrons at HC restaurants noticed nutrition information than at comparison restaurants (34.2% vs. 28.1%; OR = 1.39; p = 0.019); however, only 5% of HC restaurant patrons recalled seeing the HC symbol. HC restaurant patrons were more likely to say that their order was influenced by nutrition information (10.9% vs. 4.5%; OR = 2.96, p restaurant patrons ordered HC approved items; however, only 1% ordered a HC item and mentioned seeing the symbol in the restaurant in an unprompted recall task, and only 4% ordered a HC item and reported seeing the symbol on the item when asked directly. The HC program was associated with greater levels of noticing and influence of nutrition information, and more favourable nutrient intake; however, awareness of the HC program was very low and differences most likely reflect the type of restaurants that "self-selected" into the program.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    Highlights: • SIP characterized RDX-degrading communities under different e-accepting conditions. • Dominant RDX degradation pathways differed under different e-accepting conditions. • More complete detoxification of RDX occurred under methanogenic and sulfate-reducing conditions than under manganese(IV) and iron(III)-reducing conditions. - Abstract: This study identified microorganisms capable of using the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) or its metabolites as carbon and/or nitrogen sources under different electron-accepting conditions using 13 C and 15 N stable isotope probing (SIP). Mesocosms were constructed using groundwater and aquifer solids from an RDX-contaminated aquifer. The mesocosms received succinate as a carbon source and one of four electron acceptors (nitrate, manganese(IV), iron(III), or sulfate) or no additional electron acceptor (to stimulate methanogenesis). When RDX degradation was observed, subsamples from each mesocosm were removed and amended with 13 C 3 - or ring- 15 N 3 -, nitro- 15 N 3 -, or fully-labeled 15 N 6 -RDX, followed by additional incubation and isolation of labeled nucleic acids. A total of fifteen 16S rRNA sequences, clustering in α- and γ-Proteobacteria, Clostridia, and Actinobacteria, were detected in the 13 C-DNA fractions. A total of twenty seven sequences were derived from different 15 N-DNA fractions, with the sequences clustered in α- and γ-Proteobacteria, and Clostridia. Interestingly, sequences identified as Desulfosporosinus sp. (in the Clostridia) were not only observed to incorporate the labeled 13 C or 15 N from labeled RDX, but also were detected under each of the different electron-accepting conditions. The data suggest that 13 C- and 15 N-SIP can be used to characterize microbial communities involved in RDX biodegradation, and that the dominant pathway of RDX biodegradation may differ under different electron-accepting conditions

  8. Isotope dilution/mass spectrometry of serum cholesterol with [3,4-13C]cholesterol: proposed definitive method

    International Nuclear Information System (INIS)

    Pelletier, O.; Wright, L.A.; Breckenridge, W.C.

    1987-01-01

    We describe a new gas-chromatographic/mass-spectrometric (GC/MS) isotope-dilution method for determination of serum cholesterol. The method has been fully optimized and documented to provide the high accuracy and precision expected for a Definitive Method. In the presence of [3,4- 13 C]cholesterol, cholesteryl esters in serum are hydrolyzed under optimum conditions and the entire cholesterol pool is extracted and derivatized to silyl ethers. The cholesterol derivatives are resolved from other sterols by gas-liquid chromatography on a fused silica column, and selected ions characteristic of cholesterol and the [3,4- 13 C]cholesterol are monitored with a GC/MS quandrupole system. We estimated the cholesterol content of samples by bracketing each sample with standards of comparable cholesterol concentration that also contained the [3,4- 13 C]cholesterol. The procedure was highly reproducible (CV less than 0.5%), better accuracy and precision being obtained with [3,4- 13 C]cholesterol than with heptadeuterated cholesterol. Mean values per gram of dry serum for one serum pool assayed by this method and that of the National Bureau of Standards differed by 0.5%. We conclude that the method satisfies the criteria for a Definitive Method

  9. What is hiding behind ontogenic d13C variations in mollusk shells: New insights from scallops.

    Science.gov (United States)

    Chauvaud, L.; Lorrain, A.; Gillikin, D. P.; Thebault, J.; Paulet, Y.; Strand, O.; Blamart, D.; Guarini, J.; Clavier, J.

    2008-12-01

    We examined d13Ccalcite variations along scallop shells (Pecten maximus) sampled in Norway, France and Spain. Time series of shell calcite d13C show a consistent pattern of decreasing d13C with age. This almost linear d13C trend reflects an increasing contribution of metabolic CO2 to skeletal carbonate throughout ontogeny. We have removed this ontogenic trend to try to extract other information from our shell calcite d13C dataset. Scallops from the Bay of Brest (western Brittany, France) were then used to interpret the data as many environmental parameters were available for this site. d13Ccalcite variations were compared to d13C of dissolved inorganic carbon (DIC) and Chl a. The detrended calcite d13C profiles seem to follow a seasonal pattern, but surprisingly are inversely related to the d13C DIC and chlorophyll a concentrations measured within the water column. Theses results suggest that shell d13C variations are not controlled by isotopic variation of DIC. Since scallops eat phytoplankton and microphytobenthos cells, and, as a consequence respire organic mater largely depleted in 13C, we therefore suggest that in mollusk suspension feeders the shell d13Ccalcite might still be used to track the annual number of phytoplankton blooms when d13C values of calcite are detrended. We must consider this trend as a potential biological filter hiding precious environmental records.

  10. Effect of the nitrogen unshared electron pair on the direct /sup 13/C-/sup 13/C spin-spin coupling constant of a neighboring bond in oximes

    Energy Technology Data Exchange (ETDEWEB)

    Shcherbakov, V.V.; Krivdin, L.B.; Kalabin, G.A.; Trofimov, B.A.

    1986-11-20

    The authors have previously established that the direct /sup 13/C-/sup 13/C coupling constants are stereospecific relative to the orientation of unshared electron pairs (UEP) of nitrogen and oxygen atoms. Here they show that the nitrogen UEP produces a positive contribution to the direct /sup 13/C-/sup 13/C coupling constant of an adjacent syn-periplanar carbon-carbon bond and not to a negative contribution of the corresponding constant of the anti-periplanar bond. Thus, the observed effect is not a consequence of the interaction of the heteroatom UEP with the anti-bonding orbital of the adjacent anti-periplanar bond (n/sub o-o/* interaction) as in the case of anomeric and related effects.

  11. /sup 13/C-/sup 13/C spin-spin coupling constants in structural investigations. I. New method of determining the configuration of oximes and their derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Krivdin, L.B.; Shcherbakov, V.V.; Kalabin, G.A.

    1986-07-10

    It was shown that the direct /sup 13/C-/sup 13/C spin-spin coupling constants can be used for the unambiguous identification of the configurational isomers of oximes and their derivatives. The stereospecificity of the constants is explained by the additional contribution from the unshared electron pair of the nitrogen atom to the spin-spin coupling constant between the adjacent carbon nuclei in the cis position.

  12. /sup 13/C-/sup 13/C spin-spin coupling constants in structural investigations. V. The direct carbon-carbon coupling constants in the vinyl group

    Energy Technology Data Exchange (ETDEWEB)

    Krivdin, L.B.; Shcherbakov, V.V.; Kalabin, G.A.

    1988-03-10

    The direct spin-spin coupling constants in the vinyl group were measured in 100 mono-substituted ethylene derivatives. The inductive effect of the substituent was found to be the major factor in the variation of this constant and, in some cases, the stereospecific effect of the unshared electron pairs of heteratoms makes a significant contribution to the /sup 13/C-/sup 13/C coupling constants.

  13. delta 13C analyses of vegetable oil fatty acid components, determined by gas chromatography--combustion--isotope ratio mass spectrometry, after saponification or regiospecific hydrolysis.

    Science.gov (United States)

    Woodbury, S E; Evershed, R P; Rossell, J B

    1998-05-01

    The delta 13C values of the major fatty acids of several different commercially important vegetable oils were measured by gas chromatography--combustion--isotope ratio mass spectrometry. The delta 13C values obtained were found to fall into two distinct groups, representing the C3 and C4 plants classes from which the oils were derived. The delta 13C values of the oils were measured by continuous flow elemental isotope ratio mass spectrometry and were found to be similar to their fatty acids, with slight differences between individual fatty acids. Investigations were then made into the influence on the delta 13C values of fatty acids of the position occupied on the glycerol backbone. Pancreatic lipase was employed to selectively hydrolyse fatty acids from the 1- and 3-positions with the progress of the reaction being followed by high-temperature gas chromatography in order to determine the optimum incubation time. The 2-monoacylglycerols were then isolated by thin-layer chromatography and fatty acid methyl esters prepared. The delta 13C values obtained indicate that fatty acids from any position on the glycerol backbone are isotopically identical. Thus, whilst quantification of fatty acid composition at the 2-position and measurement of delta 13C values of oils and their major fatty acids are useful criteria in edible oil purity assessment, measurement of delta 13C values of fatty acids from the 2-position does not assist with oil purity assignments.

  14. Effect of alcohol consumption on the liver detoxication capacity as measured by [13C]methacetin- and [methyl-13C]methionine-breath tests.

    Science.gov (United States)

    Wutzke, Klaus D; Forberger, Anke; Wigger, Marianne

    2008-06-01

    The aim of this study was to investigate the hepatic microsomal and mitochondrial functions by using the 13CO2-breath test in healthy subjects either before or after the consumption of red wine. Fourteen adults received [13C]methacetin and [methyl-13C]methionine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol/kg/day together with dinner over a 10-day period. Thereafter, 13C-tracer administration was repeated under identical conditions. The 13CO2-enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage 13C-dose recovery (CPDR) after administration of [13C]methacetin and [methyl-13C]methionine either without or with red wine consumption amounted to 38.2+/-6.3 vs. 36.3+/-6.7% (p=0.363) and 9.5+/-3.3 vs. 8.8+/-2.5% (p=0.47), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the mitochondrial functions of the human liver in healthy subjects.

  15. Effect of alcohol consumption on the liver detoxication capacity as measured by [13C2]aminopyrine and L-[1-13C]phenylalanine breath tests.

    Science.gov (United States)

    Wutzke, Klaus D; Wigger, Marianne

    2009-09-01

    The aim of this study was to investigate the hepatic microsomal and cytosolic functions by using the 13CO2 breath test in healthy subjects either before or after consumption of red wine. Twelve adults received [13C2]aminopyrine and L-[1-13C]phenylalanine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol per kg per day together with dinner over a 7.5-day period on average. Thereafter, 13C-tracer administration was repeated under identical conditions. The 13CO2 enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage 13C-dose recovery after administration of [13C2]aminopyrine and L-[1-13C]phenylalanine either without or with red wine consumption amounted to 17.0+/-4.4 vs. 14.7+/-3.1% (p=0.170) and 14.0+/-2.8 vs. 11.5+/-3.9% (p=0.084), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the cytosolic function of the human liver in healthy subjects.

  16. Unexpected relationships between δ13C and wine grape performance in organic farming

    Directory of Open Access Journals (Sweden)

    Edoardo Antonio Costantino Costantini

    2013-12-01

    Significance and impact of the study: Water nutrition is crucial for wine grape performance. δ13C is a method used to assess vine water status during the growing season and to estimate vine performance. A good performance is expected at moderate stress and intermediate δ13C values. A better knowledge of the interaction between water and nutrient scarcity is needed, as it can affect the use of δ13C to predict vine performance.

  17. Needle-Age Related Variability in Nitrogen, Mobile Carbohydrates, and δ13C within Pinus koraiensis Tree Crowns

    Science.gov (United States)

    Yan, Cai-Feng; Han, Shi-Jie; Zhou, Yu-Mei; Wang, Cun-Guo; Dai, Guan-Hua; Xiao, Wen-Fa; Li, Mai-He

    2012-01-01

    For both ecologists and physiologists, foliar physioecology as a function of spatially and temporally variable environmental factors such as sunlight exposure within a tree crown is important for understanding whole tree physiology and for predicting ecosystem carbon balance and productivity. Hence, we studied concentrations of nitrogen (N), non-structural carbohydrates (NSC = soluble sugars + starch), and δ13C in different-aged needles within Pinus koraiensis tree crowns, to understand the needle age- and crown position-related physiology, in order to test the hypothesis that concentrations of N, NSC, and δ13C are needle-age and crown position dependent (more light, more photosynthesis affecting N, NSC, and δ13C), and to develop an accurate sampling strategy. The present study indicated that the 1-yr-old needles had significantly higher concentration levels of mobile carbohydrates (both on a mass and an area basis) and Narea (on an area basis), as well as NSC-N ratios, but significantly lower levels of Nmass (on a mass basis) concentration and specific leaf area (SLA), compared to the current-year needles. Azimuthal (south-facing vs. north-facing crown side) effects were found to be significant on starch [both on a mass (STmass) and an area basis (STarea)], δ13C values, and Narea, with higher levels in needles on the S-facing crown side than the N-facing crown side. Needle Nmass concentrations significantly decreased but needle STmass, STarea, and δ13C values significantly increased with increasing vertical crown levels. Our results suggest that the sun-exposed crown position related to photosynthetic activity and water availability affects starch accumulation and carbon isotope discrimination. Needle age associated with physiological activity plays an important role in determining carbon and nitrogen physiology. The present study indicates that across-scale sampling needs to carefully select tissue samples with equal age from a comparable crown position

  18. Experimental and DFT evaluation of the 1H and 13C NMR chemical shifts for calix[4]arenes

    Science.gov (United States)

    Guzzo, Rodrigo N.; Rezende, Michelle Jakeline Cunha; Kartnaller, Vinicius; Carneiro, José Walkimar de M.; Stoyanov, Stanislav R.; Costa, Leonardo Moreira da

    2018-04-01

    The density functional theory is employed to determine the efficiency of 11 exchange-correlation (XC) functionals to compute the 1H and 13C NMR chemical shifts of p-tert-butylcalix[4]arene (ptcx4, R1 = C(CH3)3) and congeners using the 6-31G(d,p) basis set. The statistical analysis shows that B3LYP, B3PW91 and PBE1PBE are the best XC functionals for the calculation of 1H chemical shifts. Moreover, the best results for the 13C chemical shifts are obtained using the LC-WPBE, M06-2X and wB97X-D functionals. The performance of these XC functionals is tested for three other calix[4]arenes: p-sulfonic acid calix[4]arene (sfxcx4 - R1 = SO3H), p-nitro-calix[4]arene (ncx4, R1 = NO2) and calix[4]arene (cx4 - R1 = H). For 1H chemical shifts B3LYP, B3PW91 and PBE1PBE yield similar results, although B3PW91 shows more consistency in the calculated error for the different structures. For 13C NMR chemical shifts, the XC functional that stood out as best is LC-WPBE. Indeed, the three functionals selected for each of 1H and 13C show good accuracy and can be used in future studies involving the prediction of 1H and 13C chemical shifts for this type of compounds.

  19. A voluntary nutrition labeling program in restaurants: Consumer awareness, use of nutrition information, and food selection

    Directory of Open Access Journals (Sweden)

    Christine M. White

    2016-12-01

    Full Text Available Health Check (HC was a voluntary nutrition labeling program developed by the Heart and Stroke Foundation of Canada as a guide to help consumers choose healthy foods. Items meeting nutrient criteria were identified with a HC symbol. This study examined the impact of the program on differences in consumer awareness and use of nutritional information in restaurants. Exit surveys were conducted with 1126 patrons outside four HC and four comparison restaurants in Ontario, Canada (2013. Surveys assessed participant noticing of nutrition information, influence of nutrition information on menu selection, and nutrient intake. Significantly more patrons at HC restaurants noticed nutrition information than at comparison restaurants (34.2% vs. 28.1%; OR = 1.39; p = 0.019; however, only 5% of HC restaurant patrons recalled seeing the HC symbol. HC restaurant patrons were more likely to say that their order was influenced by nutrition information (10.9% vs. 4.5%; OR = 2.96, p < 0.001; and consumed less saturated fat and carbohydrates, and more protein and fibre (p < 0.05. Approximately 15% of HC restaurant patrons ordered HC approved items; however, only 1% ordered a HC item and mentioned seeing the symbol in the restaurant in an unprompted recall task, and only 4% ordered a HC item and reported seeing the symbol on the item when asked directly. The HC program was associated with greater levels of noticing and influence of nutrition information, and more favourable nutrient intake; however, awareness of the HC program was very low and differences most likely reflect the type of restaurants that “self-selected” into the program.

  20. Ecosystem differences in the trophic enrichment of 13C in aquatic food webs

    International Nuclear Information System (INIS)

    France, R.L.; Peters, R.H.

    1997-01-01

    Data from 35 published studies were collated to examine patterns in the trophic enrichment of 13 C of consumers. Because both δ 13 C and δ 14 N vary systematically across ecosystems, it was necessary to standardize for such differences before combining data from numerous sources. Relationships of these measures of ecosystem-standardized δ 13 C to ecosystem-standardized trophic position (Δδ 15 N) for freshwater, estuarine, coastal, and open-ocean and for all aquatic ecosystems yielded regression equations of low predictive capability (average of 20% explained variance in δ 13 C). However, differences were observed in the slopes between δ 13 C and standardized trophic position when data were examined study-specifically: the average trophic fractionation of 13 C was found to increase from +0.2micron for freshwater to +0.5micron for estuarine to +0.8micron for coastal, and to +1.1micron for open-ocean food webs. This ecosystem-specific gradient in 13 C enrichment for consumers supports previous findings of a similar continuum existing for zooplankton - particulate organic matter differences in δ 13 C. Possible mechanisms to explain these ecosystem-specific patterns in 13 C enrichment may be related to the relative importance of detritus, heterotrophic respiration, partial reliance on alternative food sources, and lipid influences in the different ecosystems. (author)

  1. Autoradiography and density gradient separation of technetium-99m-Exametazime (HMPAO) labelled leucocytes reveals selectivity for eosinophils

    Energy Technology Data Exchange (ETDEWEB)

    Puncher, M.R.B. [Biological Lab., Univ. of Kent, Canterbury (United Kingdom); Blower, P.J. [Nuclear Medicine Dept., Kent and Canterbury Hospital (United Kingdom)

    1994-11-01

    Technetium-99m-Exametazime (HMPAO) is widely used for radiolabelling leucocytes for localization of infection. The subcellular distribution of radionuclide in the labelled cells and the distribution of radioactivity among the leucocyte population are incompletely understood. Frozen section autoradiography was used to determine quantitatively the distribution of {sup 99m}Tc in leucocytes labelled with {sup 99m}Tc-Exametazime. Sections of rapidly frozen suspensions of labelled leucocytes in plasma were autoradiographed on Ilford K2 emulsion and stained with haematoxylin and eosin. Neutrophils, eosinophils and mononuclear cells were separated by Percoll density gradient centrifugation. Cell nuclei were isolated by a rapid cell-breakage and fractionation method. In a typical experiment mean grain densities [grains/100 {mu}m{sup 2} (ESD)] over cells were: eosinophils 31.2 (18.4), neutrophils 3.5 (3.5), mononuclear cells 4.2 (5.1). Mean grain numbers per cell (ESD) were: eosinophils 13 (6.8), neutrophils 1.3 (1.3), mononuclear cells 1.1 (1.3). These findings were confirmed by separation of labelled leucocytes on discontinuous density gradients. In four separation experiments, the mean activity-per-cell ratio for eosinophils to neutrophils was 10.1 (4.8):1, and for eosinophils to mononuclear cells, 14.1 (6.7):1. The subcellular distribution of the label was investigated using image analysis of autoradiographs and cell fractionation. This revealed no selectivity for nuclear or extranuclear compartments. It may be concluded that {sup 99m}Tc-Exametazime has strong selectivity for eosinophils over other leucocytes but no selectivity for nuclear/cytoplasmic compartments. (orig.)

  2. Selective retrograde labeling of cholinergic neurons with [3H]choline

    International Nuclear Information System (INIS)

    Bagnoli, P.; Beaudet, A.; Stella, M.; Cuenod, M.

    1981-01-01

    Evidence is presented which is consistent with a specific retrograde labeling of cholinergic neurons following [ 3 H]choline application in their zone of termination. [ 3 H]Choline injection in the rat hippocampus leads to perikaryal retrograde labeling in the ipsilateral medial septal nuclease and nucleus of the diagonal band, thus delineating an established cholinergic pathway, while only diffuse presumably anterograde labeling was observed in the lateral septum, the entorhinal cortex, and the opposite hippocampus. After [ 3 H]choline injection in the pigeon visual Wulst, only the ipsilateral thalamic relay, of all inputs, showed similar perikaryal retrograde labeling, an observation supporting the suggestion that at least some thalamo-Wulst neurons are cholinergic

  3. Creating 13C- and 15N-enriched tree leaf litter for decomposition experiments

    Science.gov (United States)

    Szlavecz, K. A.; Pitz, S.; Chang, C.; Bernard, M.

    2013-12-01

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

  4. Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: Potential for NMR structure determination of large proteins

    International Nuclear Information System (INIS)

    Kelly, Mark J.S.; Krieger, Cornelia; Ball, Linda J.; Yu Yihua; Richter, Gerald; Schmieder, Peter; Bacher, Adelbert; Oschkinat, Hartmut

    1999-01-01

    NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  6. Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate

    Science.gov (United States)

    Nelson, Sarah J.; Kurhanewicz, John; Vigneron, Daniel B.; Larson, Peder E. Z.; Harzstark, Andrea L.; Ferrone, Marcus; van Criekinge, Mark; Chang, Jose W.; Bok, Robert; Park, Ilwoo; Reed, Galen; Carvajal, Lucas; Small, Eric J.; Munster, Pamela; Weinberg, Vivian K.; Ardenkjaer-Larsen, Jan Henrik; Chen, Albert P.; Hurd, Ralph E.; Odegardstuen, Liv-Ingrid; Robb, Fraser J.; Tropp, James; Murray, Jonathan A.

    2014-01-01

    This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-13C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-fold enhancement in signal relative to conventional magnetic resonance (MR) imaging. When combined with the rapid acquisition of in vivo 13C MR data, it is possible to evaluate the distribution of agents such as [1-13C]pyruvate and its metabolic products lactate, alanine, and bicarbonate in a matter of seconds. Preclinical studies in cancer models have detected elevated levels of hyperpolarized [1-13C]lactate in tumor, with the ratio of [1-13C]lactate/[1-13C]pyruvate being increased in high-grade tumors and decreased after successful treatment. Translation of this technology into humans was achieved by modifying the instrument that generates the hyperpolarized agent, constructing specialized radio frequency coils to detect 13C nuclei, and developing new pulse sequences to efficiently capture the signal. The study population comprised patients with biopsy-proven prostate cancer, with 31 subjects being injected with hyperpolarized [1-13C]pyruvate. The median time to deliver the agent was 66 s, and uptake was observed about 20 s after injection. No dose-limiting toxicities were observed, and the highest dose (0.43 ml/kg of 230 mM agent) gave the best signal-to-noise ratio for hyperpolarized [1-13C]pyruvate. The results were extremely promising in not only confirming the safety of the agent but also showing elevated [1-13C]lactate/[1-13C]pyruvate in regions of biopsy-proven cancer. These findings will be valuable for noninvasive cancer diagnosis and treatment monitoring in future clinical trials. PMID:23946197

  7. NMR study of conjugation effects. 15. /sup 13/C-/sup 13/C spin-spin coupling constants in phenylalkyl ether

    Energy Technology Data Exchange (ETDEWEB)

    Kalabin, G.A. (Siberian Branch of the Academy of Sciences of the USSR); Krivdin, L.B.; Trofimov, B.A.

    1982-07-20

    In order to elucidate the /sup 13/C-/sup 13/-C SSCC (spin-spin coupling constants) segment with the electronic excitations induced by the R group, a series of phenyl alkyl ethers, PhOAlk, where Alk = Me(I), Et(II), i-Pr(III), and t-Bu(IV), were studied. This series was chosen because in studying the /sup 13/C CS in monosubstituted benzenes it was observed that the intensity of the ..pi..-electron interaction of the unshared electron pairs of oxygen with the ..pi.. system of the benzene ring was practically the same in some compounds, but increased by 30% in others. This is related to the fact that the latter is characterized by an average noncoplanar conformation, with a dihedral angle between the benzene-ring plane and the C-O-C bond of approx. 45/sup 0/, whereas some compounds have an angle < 20/sup 0/. The reason for the difference is significant steric interaction of the alkyl hydrogens with the o-position of the ring. Thus, consideration of the /sup 13/C-/sup 13/C SSCC of a series of quite similar compounds, especially when compared to the whole set of such SSCC for other monosubstituted benzenes, shows that their relationship to the structure of the substituent R is extremely complex.

  8. Measurement of the 13C/12C ratio of soil-plant individual sugars by gas chromatography/combustion/isotope-ratio mass spectrometry of silylated derivatives.

    Science.gov (United States)

    Derrien, Delphine; Balesdent, Jérôme; Marol, Christine; Santaella, Catherine

    2003-01-01

    Carbohydrate is an important pool in the terrestrial carbon cycle. The potential offered by natural and artificial 13C-labelling techniques should therefore be applied to the investigation of the dynamics of individual sugars in soils. For this reason, we evaluated the method of 13C sugar analysis by gas chromatography/combustion/isotope-ratio mass spectrometry (GC/C/IRMS) after hydrolysis and direct trimethylsilylation. Trimethylsilylation involved the addition of several carbon atoms per sugar. These atoms have to be taken into account in the estimation of the carbon isotope ratio. The analysis of standard and natural pentoses and hexoses of known 13C enrichments revealed that the number of analysed added carbon atoms was less than expected from stoichiometry. This was attributed to incomplete derivatization and/or incomplete oxidation of methylsilyl carbon before IRMS. Using a calibration of the number of analysed added carbon atoms, the isotope excess of enriched samples could be determined with a relative error close to 5%. Concerning the determination of natural abundances by GC/C/IRMS, we could measure the delta 13C of standard C3- and C4-derived sugars with an accuracy of +/-1.5 per thousand using the previous calibration. We were able to apply this technique to plant-soil systems labelled by pulse-chase of 13CO2, revealing the nature and dynamics of sugars in the plant rhizosphere. Copyright 2003 John Wiley & Sons, Ltd.

  9. Potential effect of physical activity based menu labels on the calorie content of selected fast food meals.

    Science.gov (United States)

    Dowray, Sunaina; Swartz, Jonas J; Braxton, Danielle; Viera, Anthony J

    2013-03-01

    In this study we examined the effect of physical activity based labels on the calorie content of meals selected from a sample fast food menu. Using a web-based survey, participants were randomly assigned to one of four menus which differed only in their labeling schemes (n=802): (1) a menu with no nutritional information, (2) a menu with calorie information, (3) a menu with calorie information and minutes to walk to burn those calories, or (4) a menu with calorie information and miles to walk to burn those calories. There was a significant difference in the mean number of calories ordered based on menu type (p=0.02), with an average of 1020 calories ordered from a menu with no nutritional information, 927 calories ordered from a menu with only calorie information, 916 calories ordered from a menu with both calorie information and minutes to walk to burn those calories, and 826 calories ordered from the menu with calorie information and the number of miles to walk to burn those calories. The menu with calories and the number of miles to walk to burn those calories appeared the most effective in influencing the selection of lower calorie meals (p=0.0007) when compared to the menu with no nutritional information provided. The majority of participants (82%) reported a preference for physical activity based menu labels over labels with calorie information alone and no nutritional information. Whether these labels are effective in real-life scenarios remains to be tested. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. The synthesis of 13C-bilirubin and its use in the validation of bilirubin kinetic studies in rats

    International Nuclear Information System (INIS)

    Sturrock, E.D.

    1994-04-01

    The total synthesis of [10- 13 C] bilirubin IXα], the principal waste product of haem degradation, is described. Site specific labelling was accomplished by the Vilsmeier formulation of one of the dipyrrolic fragments using [1- 13 C] dimethylformamide. The penultimate dehydrohalogenation reaction was complicated by a competing elimination reaction which yielded a bridged biliverdin derivative. The base catalysed reaction affords a novel [10- 13 C]-8,12-bis(2-methoxycarbonylethyl)-7,13,17-trimethyl-2,18-propano-3-vinylbilin-1,19(21H,24H)-dione in which the 2 and 18 positions of the macrocycle are bridged with a propano tether, the structure has been established using single crystal X-ray and 1 H nuclear Overhauser effect studies. [ 14 C]bilirubin was prepared, bio synthetically, using [ 14 C]aminolevulinic acid. Bilirubin kinetics in 4 rats were measured by the analysis of the plasma disappearance of [ 14 C]bilirubin in a two-compartment model. The plasma half-life of the first and second exponentials were 1.97 and 32.8 minutes respectively. The data were used to determine model independent parameters k 12 , k 21 , and k 20 . In the proposed model, plasma unconjugated bilirubin exchanges with a hepatic unconjugated bilirubin pool. Bilirubin is eliminated from the system via the proposed hepatic pool. These studies provide an analysis of the kinetics of unconjugated bilirubin in rates and are intended to serve as a reference point for studies using a stable isotope of bilirubin. The plasma disappearance of [10- 13 C]bilirubin IXα in three rats was studied using mass spectrometry to measure the bilirubin δ 13 C. Validation of the experimental procedure in terms of range and reproducibility of the detection method was carried out. The half lives of the initial and terminal exponentials were 2.27±2.5 and 22.8±12.9 minutes. Despite the large 95% confidence limits calculated for these clearance curves they serve as an important foundation for future bilirubin kinetic

  11. Compartmental and noncompartmental modeling of 13C-lycopene absorption, isomerization, and distribution kinetics in healthy adults123

    Science.gov (United States)

    Moran, Nancy E; Cichon, Morgan J;