Improved transfer efficiencies in radio-frequency-driven recoupling solid-state NMR by adiabatic sweep through the dipolar recoupling condition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Straasø, Lasse A.; Shankar, Ravi; Nielsen, Niels Chr.

The homonuclear radio-frequency driven recoupling (RFDR) experiment is commonly used in solid-state NMR spectroscopy to gain insight into the structure of biological samples due to its ease of implementation, stability towards fluctuations/missetting of radio-frequency (rf) field strength, and in general low rf requirements. A theoretical operator-based Floquet description is presented to appreciate the effect of having a temporal displacement of the π-pulses in the RFDR experiment. From this description, we demonstrate improved transfer efficiency for the RFDR experiment by generating an adiabatic passage through the zero-quantum recoupling condition. We have compared the performances of RFDR and the improved sequence tomore » mediate efficient {sup 13}CO to {sup 13}C{sub α} polarization transfer for uniformly {sup 13}C,{sup 15}N-labeled glycine and for the fibril forming peptide SNNFGAILSS (one-letter amino acid codes) uniformly {sup 13}C,{sup 15}N-labeled at the FGAIL residues. Using numerically optimized sweeps, we get experimental gains of approximately 20% for glycine where numerical simulations predict an improvement of 25% relative to the standard implementation. For the fibril forming peptide, using the same sweep parameters as found for glycine, we have gains in the order of 10%–20% depending on the spectral regions of interest.« less

Structure of the Putative 32 kDa Myrosinase Binding Protein from Arabidopsis (At3g16450.1) Determined by SAIL-NMR

PubMed Central

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira Mei; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, JunGoo; Güntert, Peter; Aceti, David J.; Markley, John L.; Kainosho, Masatsune

2009-01-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme, myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniformly 13C/15N labeling methods, we used our stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected with the SAIL-protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at the low concentration (0.2 mM) of the protein product. We collected additional NOESY data and solved the three-dimensional structure with the CYANA software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent, but similar, lectin-fold domains composed of three β-sheets. PMID:19021763

Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR.

PubMed

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira M; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, Jungoo; Güntert, Peter; Aceti, David J; Markley, John L; Kainosho, Masatsune

2008-12-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

Backbone resonance assignments of the PRYSPRY domain of TRIM25.

PubMed

Kong, Chen; Penumutchu, Srinivasa R; Hung, Kuo-Wei; Huang, Huiying; Lin, Tianwei; Yu, Chin

2015-10-01

TRIM25 is a member of the tripartite motif (TRIM) family and has been implicated in the regulation of innate immune signaling via the RIG-I (retinoic acid-inducible gene-I) pathway for antiviral defense. As the essential first step towards the structural and functional characterization of the TRIM25/RIG-I interaction, the backbone resonance of the PRYSPRY domain of TRIM25 is assigned here based on triple-resonance experiments using uniformly [(2)H, (13)C, (15)N]-labeled protein.

C4'/H4' selective, non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of (13)C-labeled RNAs.

PubMed

Saxena, Saurabh; Stanek, Jan; Cevec, Mirko; Plavec, Janez; Koźmiński, Wiktor

2014-11-01

A through bond, C4'/H4' selective, "out and stay" type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4'(i)-C4'(i)-C4'(i-1)-H4'(i-1) correlations. The (31)P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4' dimension. The experiment fully utilizes (13)C-labeling of RNA by inclusion of two C4' evolution periods. An additional evolution of H4' is included to further enhance peak resolution. Band selective (13)C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4'-C3' and C4'-C5' homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully (13)C, (15)N-labeled 34-nt hairpin RNA comprising typical structure elements.

Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy.

PubMed

Nestor, Gustav; Anderson, Taigh; Oscarson, Stefan; Gronenborn, Angela M

2017-05-03

NMR of a uniformly 13 C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13 C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13 C spectral dispersion of 13 C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

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

PubMed

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

2017-05-01

In this paper, the effect of isotope-labeled analogs on the liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) measurement was evaluated based on the comparison research of electrospray ionization responses (ESI) and matrix effect of melamine, 13 C 3 -melamine, 13 C 3 + 15 N 3 -melamine, and 15 N 3 -melamine. The isotope-labeled melamines had similar ionization efficiency with melamine in the electrospray ionization source, but the intensity of corresponding quantitative fragment ions had distinctive differences. Based on the density functional theory at the B3LYP/6-311+G** level, this phenomenon was explained very well. The rare cleavage pathways of melamine, which just could be exactly identified by 15 N-labeled melamines, resulted in the difference of quantitative fragment ions between 15 N-labeled melamines and melamine. The interaction of ESI response between melamine and isotope-labeled melamines was investigated using MRM monitor mode. 15 N-labeled melamine had significant ion inter-suppression effect on melamine, while 13 C-labeled melamine had little influence on melamine. Finally, the influence of different isotope-labeled melamines on the LC-IDMS result was evaluated using the IDMS correction factor (θ). Taking the determination of melamine in milk powder as an example, the matrix effects of different isotope-labeled melamines and melamine had notable difference and the impact of this difference on the measurement results depended on the concentrations of analyte and matrix solution. It was worth noting that 15 N 3 -melamine exhibited significant ion suppression to melamine in matrix solution. The deviation of the results from IDMS method might reach 59% using 15 N 3 -melamine as internal standard in special matrix solution. Graphical Abstract The comparison of ESI responses of melamine, 13 C 3 -melamine, 13 C 3 + 15 N 3 -melamine and 15 N 3 -melamine.

NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vourtsis, Dionysios J.; Chasapis, Christos T.; Pairas, George

2014-07-18

Highlights: • A polypeptide, N-ALF{sub 233}, was overexpressed in E. coli and successfully isolated. • We produced {sup 2}H/{sup 15}N/{sup 13}C labeled protein samples. • Amino acid selective approaches were applied. • We acquired several heteronuclear NMR spectra, to complete the backbone assignment. • Prediction of the secondary structure was performed. - Abstract: NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successfulmore » overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.« less

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.

PubMed

Dalluge, Joseph J; McCurtain, Jennifer L; Gilbertsen, Adam J; Kalstabakken, Kyle A; Williams, Bryan J

2015-07-01

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1% (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa

PubMed Central

McCurtain, Jennifer L.; Gilbertsen, Adam J.; Kalstabakken, Kyle A.; Williams, Bryan J.

2018-01-01

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled 13C5,15N4-agmatine (synthesized by decarboxylation of uniformly labeled 13C6,15N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,l,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1 % (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients. PMID:25957842

Endogenous biosynthesis of nitrate and its role in vivo nitrosation. [Mustela putorius furo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dull, B.J.

1985-01-01

The ferret (Mustela putorius furo) was chosen as a model animal for the metabolism of nitrate. The net biosynthesis of nitrate in the ferret was demonstrated to be 8.89-10.3 mcmole/kg/day. Nitrate balance studied indicate that, because of metabolism, excretion of nitrate was lower than ingestion which oral doses were higher than 6.3 mcmole/day. At nitrate ingestions levels of less than 6.3 mcmole/day, excretion exceeded intake. Studies with 15-N labelled nitrate indicated that only 36% of the oral dose was recovered as 15-N nitrate from urine. Oral doses of 15-N labelled ammonia resulted in the incorporation of the 15-N label intomore » nitrate in urine and feces. This demonstrated that ammonia can serve as a precursor to biosynthesized nitrate. Oral dosing of ferrets with N-nitrosoproline results in 94.7% recovery in the urine within 48 hr. 15-N labelled N-nitrosoproline could not be detected in the urine at anytime during a ten day feeding study with 15-N labelled nitrate. 14-N labelled N-nitrosoproline was detected throughout. As 14-N N-nitrosoproline could not be detected in the diet, it would appear that excretion of 14-N labelled N-nitrosoproline must be arising from an in vivo nitrosation site other than the stomach.« less

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

PubMed

Lanquar, Viviane; Kuhn, Lauriane; Lelièvre, Françoise; Khafif, Mehdi; Espagne, Christelle; Bruley, Christophe; Barbier-Brygoo, Hélène; Garin, Jérôme; Thomine, Sébastien

2007-03-01

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

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

NASA Astrophysics Data System (ADS)

Kleineidam, Kristina; Müller, Christoph

2017-04-01

Mineralisation is a key N transformation process supplying reactive nitrogen (N) to terrestrial ecosystems. The various soil organic matter fractions contribute to the total mineralisation according to their turnover characteristic. However, the exact mechanism and the gross dynamics of the various processes are not well understood. In this study we investigated the mineralisation-immobilisation dynamics in a grassland soil by a combined field-laboratory study. Eighteen microplots were established at a field site receiving 50 kg N ha-1 as ammonium nitrate. In nine (3 x 3) respective plots the ammonium, or the nitrate, or both moieties were 15N labelled at 60 atom%. Previous studies with this soil showed that rapid turnover occurred and available N would partly be immobilised by the microbial biomass increasing the 15N label of the soil organic nitrogen pool in the field. After one year, soil samples were taken from the 15N treated and the so far non-labelled plots and examined in a laboratory study (for details of the setup see: Müller et al., 2004). While the previously differentially 15N labelled field soils were now supplied with unlabelled ammonium nitrate, the previously unlabelled soils were now treated with either 15N labelled ammonium nitrate similar to the 15N treatments established in the field, resulting in six different 15N treatments in total. The incubation study was carried out over a two week period and data were analysed with the Ntrace model to quantify the simultaneously occurring gross N transformations while optimizing a single parameter set for all six treatments. Thus, the appearance of 15N from the previously labelled soils and the dilution of the 15N in the recently labelled treatments were assumed to be driven by the same processes and activities and were used to constrain the 15N tracing model. This approach allowed us to estimate the individual gross N transformation rates with a much higher accuracy than if only a common triple labelling approach had been used. Here we present detailed gross N turnover dynamics and an improved conceptual model for the mineralisation-immobilisation dynamics in grassland soil. Literature cited Müller, C., Stevens, R.J., Laughlin, R.J., 2004. A 15N tracing model to analyse N transformations in old grassland soil. Soil Biology & Biochemistry 36, 619-632.

Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

PubMed

Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

2015-06-01

Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

Amino acid selective unlabeling for sequence specific resonance assignments in proteins

PubMed Central

Krishnarjuna, B.; Jaipuria, Garima; Thakur, Anushikha

2010-01-01

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling’ or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly 13C/15N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {12COi–15Ni+1}-filtered HSQC, which aids in linking the 1HN/15N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i − 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to 2H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of 14N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies. Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9459-z) contains supplementary material, which is available to authorized users. PMID:21153044

Refining cotton-wick method for 15N plant labelling.

NASA Astrophysics Data System (ADS)

Fustec, Joëlle; Mahieu, Stéphanie

2010-05-01

The symbiosis Fabaceae/Rhizobiaceae plays a critical role in the nitrogen cycle. It gives the plant the ability to fix high amounts of atmospheric N. A part of this N can be transferred to the soil via rhizodeposition. The contribution of Fabaceae to the soil N pool is difficult to measure, since it is necessary for assessing N benefits for other crops, for soil biological activity, and for reducing water pollution in sustainable agriculture (Fustec, 2009). The aim of this study was to test and improve the reliability of the 15N cotton-wick method for measuring the soil N derived from plant rhizodeposition (Mahieu et al., 2007). The effects of the concentration of the 15N-urea labelling solution and of the feeding frequency (continuous or pulses) on the assessment of nitrogen rhizodeposition were studied in two greenhouse experiments using the field pea (Pisum sativum L.) and the non-nodulating isoline P2. The plant parts and the soil were prepared for 15N:14N measurements for assessing N rhizodeposition (Mahieu et al., 2009). The fraction of plants' belowground nitrogen allocated to rhizodeposition in both Frisson pea and P2 was 20 to more than 50% higher when plants were labelled continuously than when they were labelled using fortnightly pulses. Our results suggested that when 15N root enrichment was high, nitrogen rhizodeposition was underestimated only for plants that were 15N-fed by fortnightly pulses, and not in plants 15N-fed continuously. This phenomenon was especially observed for plants relying on symbiotic N fixation for N acquisition; it may be linked to the concentration of the labelling solution. In conclusion, N rhizodeposition assessment was strongly influenced by the 15N-feeding frequency and the concentration of the labelling solution. The estimation of N rhizodeposition was more reliable when plants were labelled continuously with a dilute solution of 15N urea. Fustec et al. 2009. Agron. Sustain. Dev., DOI 10.1051/agro/2009003, in press. Mahieu et al. 2007. Plant Soil 295, 193-205. Mahieu et al. 2009. Soil Biol. Biochem. 41, 2236-2243.

Extracting latent brain states--Towards true labels in cognitive neuroscience experiments.

PubMed

Porbadnigk, Anne K; Görnitz, Nico; Sannelli, Claudia; Binder, Alexander; Braun, Mikio; Kloft, Marius; Müller, Klaus-Robert

2015-10-15

Neuroscientific data is typically analyzed based on the behavioral response of the participant. However, the errors made may or may not be in line with the neural processing. In particular in experiments with time pressure or studies where the threshold of perception is measured, the error distribution deviates from uniformity due to the structure in the underlying experimental set-up. When we base our analysis on the behavioral labels as usually done, then we ignore this problem of systematic and structured (non-uniform) label noise and are likely to arrive at wrong conclusions in our data analysis. This paper contributes a remedy to this important scenario: we present a novel approach for a) measuring label noise and b) removing structured label noise. We demonstrate its usefulness for EEG data analysis using a standard d2 test for visual attention (N=20 participants). Copyright © 2015 Elsevier Inc. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

Automated NMR structure determination of stereo-array isotope labeled ubiquitin from minimal sets of spectra using the SAIL-FLYA system.

PubMed

Ikeya, Teppei; Takeda, Mitsuhiro; Yoshida, Hitoshi; Terauchi, Tsutomu; Jee, Jun-Goo; Kainosho, Masatsune; Güntert, Peter

2009-08-01

Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly 13C/15N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional "through-bond" spectrum (and 2D HSQC spectra) in addition to the 13C-edited and 15N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods.

The "Speedy" Synthesis of Atom-Specific (15)N Imino/Amido-Labeled RNA.

PubMed

Neuner, Sandro; Santner, Tobias; Kreutz, Christoph; Micura, Ronald

2015-08-10

Although numerous reports on the synthesis of atom-specific (15)N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of (15)N(1) adenosine and (15)N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve (15)N(3) uridine and (15)N(3) cytidine amidites in order to tap full potential of (1)H/(15)N/(15)N-COSY experiments for directly monitoring individual Watson-Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered. © 2015 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

Kreutz, Christoph; Micura, Ronald

2016-01-01

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

Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.

PubMed

Gupta, Sebanti; Tycko, Robert

2018-02-01

Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716-730, 2010; J Mol Biol 426:1109-1127, 2014; J Biol Chem 291:13098-13112, 2016; J Am Chem Soc 138:8538-8546, 2016; J Am Chem Soc 138:12029-12032, 2016; J Am Chem Soc 134:6455-6466, 2012; J Am Chem Soc 132:1976-1987, 2010; J Am Chem Soc 135:17793-17803, 2013; Proc Natl Acad Sci USA 112:14617-14622, 2015; J Am Chem Soc 138:14066-14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly 15 N, 13 C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.

[Dynamics of amino acid and protein metabolism of laying hens after administration of 15N-labeled wheat protein. 5. Incorporation of 15N into the blood fraction and its amino acids].

PubMed

Gruhn, K; Zander, R; Kirchner, E

1987-09-01

12 colostomized laying hens which received 15N labelled wheat over 4 days were butchered 12 h, 36 h, 60 h and 108 h (3 animals each) after the last 15N application. The intake of 15N excess (15N') from the wheat amounted to 540 mg 15N' during the application period. The 15N' in the blood plasma decreased after the last 15N' application from 0.76 atom-% to 0.55 atom-% after 108 h, the labelling of the corpuscular components at the same measuring points increased from 0.28 to 0.50 atom-% 15N'. 96.6% of the plasma 15N' and 93.8% of that in the corpuscles is precipitable in trichloric acetic acid. The atom-% 15N' of histidine in the total blood remained unchanged in dependence on the butchering time. The 15N amount in lysine and arginine and that in the non-basic amino acids decreased inconsiderably in the period between 12 h and 108 h after the last 15N' wheat feeding.

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

PubMed

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.

Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A.

PubMed

Daughdrill, Gary W; Buchko, Garry W; Botuyan, Maria V; Arrowsmith, Cheryl; Wold, Marc S; Kennedy, Michael A; Lowry, David F

2003-07-15

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA70(1-326)), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the (1)H-(15)N correlation spectrum of uniformly (15)N-labeled hRPA70(1-326) after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA70(1-326) fragment. The hRPA70(1-326) residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA70(1-326) suggests that a competitive binding mechanism mediates the formation of the RPA-XPA complex. To determine whether a ternary complex could form between hRPA70(1-326), XPA-MBD and ssDNA, a (1)H-(15)N correlation spectrum was acquired for uniformly (15)N-labeled hRPA70(1-326) after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA70(1-326) in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA70(1-326) suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA70(1-326) may be modulated by ssDNA.

Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A

PubMed Central

Daughdrill, Gary W.; Buchko, Garry W.; Botuyan, Maria V.; Arrowsmith, Cheryl; Wold, Marc S.; Kennedy, Michael A.; Lowry, David F.

2003-01-01

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1–326 (hRPA701–326), and a fragment of the human XPA protein, residues 98–219 (XPA-MBD). Intensity changes were observed for amide resonances in the 1H–15N correlation spectrum of uniformly 15N-labeled hRPA701–326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA701–326 fragment. The hRPA701–326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA701–326 suggests that a competitive binding mechanism mediates the formation of the RPA–XPA complex. To determine whether a ternary complex could form between hRPA701–326, XPA-MBD and ssDNA, a 1H–15N correlation spectrum was acquired for uniformly 15N-labeled hRPA701–326 after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA701–326 in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA701–326 suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA701–326 may be modulated by ssDNA. PMID:12853635

Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine

PubMed Central

2005-01-01

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol·min−1·(mg of protein)−1. Experiments with an increasing concentration (0– 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol·min−1·(mg of protein)−1 for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol·min−1·g−1) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway. PMID:15656789

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

NASA Technical Reports Server (NTRS)

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

1991-01-01

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

Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

PubMed

Sugiki, Toshihiko; Furuita, Kyoko; Fujiwara, Toshimichi; Kojima, Chojiro

2018-06-20

Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective 15 N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl 13 C atoms experience less isotope scrambling than the main-chain 15 N atoms do. However, little is known about the side-chain 13 C atoms. Here, the 13 C scrambling profiles of the Cα and side-chain carbons were investigated for 15 N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in 13 Cα and 13 C side-chain labeling than in 15 N labeling. We utilized this reduced scrambling-prone character of 13 C as a simple and efficient method for amino acid selective 13 C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the 13 C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. 1 H- 15 N heteronuclear single-quantum coherence signals of the 15 N scrambling-prone amino acid were also easily filtered using 15 N-{ 13 Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures.

PubMed

Mesnard, F; Azaroual, N; Marty, D; Fliniaux, M A; Robins, R J; Vermeersch, G; Monti, J P

2000-02-01

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3. By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional 15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid gamma-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into gamma-aminobutyric acid, N-acetylputrescine. The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen.

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

PubMed Central

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

2011-01-01

Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full 15N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform 15N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO2 and external acetate into protein biosynthesis, which also correlated with a rapid, but fully reversible cell cycle arrest after onset of stress. Our approach opens up new perspectives for plant systems biology and provides novel insights into plant stress acclimation. PMID:21610104

Progress in proton-detected solid-state NMR (SSNMR): Super-fast 2D SSNMR collection for nano-mole-scale proteins

NASA Astrophysics Data System (ADS)

Ishii, Yoshitaka; Wickramasinghe, Ayesha; Matsuda, Isamu; Endo, Yuki; Ishii, Yuji; Nishiyama, Yusuke; Nemoto, Takahiro; Kamihara, Takayuki

2018-01-01

Proton-detected solid-state NMR (SSNMR) spectroscopy has attracted much attention due to its excellent sensitivity and effectiveness in the analysis of trace amounts of amyloid proteins and other important biological systems. In this perspective article, we present the recent sensitivity limit of 1H-detected SSNMR using "ultra-fast" magic-angle spinning (MAS) at a spinning rate (νR) of 80-100 kHz. It was demonstrated that the high sensitivity of 1H-detected SSNMR at νR of 100 kHz and fast recycling using the paramagnetic-assisted condensed data collection (PACC) approach permitted "super-fast" collection of 1H-detected 2D protein SSNMR. A 1H-detected 2D 1H-15N correlation SSNMR spectrum for ∼27 nmol of a uniformly 13C- and 15N-labeled GB1 protein sample in microcrystalline form was acquired in only 9 s with 50% non-uniform sampling and short recycle delays of 100 ms. Additional data suggests that it is now feasible to detect as little as 1 nmol of the protein in 5.9 h by 1H-detected 2D 1H-15N SSNMR at a nominal signal-to-noise ratio of five. The demonstrated sensitivity is comparable to that of modern solution protein NMR. Moreover, this article summarizes the influence of ultra-fast MAS and 1H-detection on the spectral resolution and sensitivity of protein SSNMR. Recent progress in signal assignment and structural elucidation by 1H-detected protein SSNMR is outlined with both theoretical and experimental aspects.

Proton assisted recoupling and protein structure determination

NASA Astrophysics Data System (ADS)

de Paëpe, Gaël; Lewandowski, Józef R.; Loquet, Antoine; Böckmann, Anja; Griffin, Robert G.

2008-12-01

We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between 13C or 15N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between 1H-13C (or 1H-15N) couplings to mediate zero quantum 13C-13C (or 15N-15N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C1+/-C2-/+HZ for 13C-13C recoupling experiments (or N1+/-N2-/+HZ for 15N-15N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with 13C-13C spectra of uniformly 13C, 15N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2×10.4 kDa). The spectra, which were acquired at high MAS frequencies (ωr2π>20 kHz) and magnetic fields (750-900 MHz 1H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A˚) 13C-13C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein.

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

PubMed

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

2016-01-01

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

Comparison of three 15N methods to correct for microbial contamination when assessing in situ protein degradability of fresh forages.

PubMed

Kamoun, M; Ammar, H; Théwis, A; Beckers, Y; France, J; López, S

2014-11-01

The use of stable (15)N as a marker to determine microbial contamination in nylon bag incubation residues to estimate protein degradability was investigated. Three methods using (15)N were compared: (15)N-labeled forage (dilution method, LF), (15)N enrichment of rumen solids-associated bacteria (SAB), and (15)N enrichment of rumen liquid-associated bacteria (LAB). Herbage from forages differing in protein and fiber contents (early-cut Italian ryegrass, late-cut Italian ryegrass, and red clover) were freeze-dried and ground and then incubated in situ in the rumen of 3 steers for 3, 6, 12, 24, and 48 h using the nylon bag technique. The (15)N-labeled forages were obtained by fertilizing the plots where herbage was grown with (15)NH4 (15)NO3. Unlabeled forages (obtained from plots fertilized with NH4NO3) were incubated at the same time that ((15)NH4)2SO4 was continuously infused into the rumen of the steers, and then pellets of labeled SAB and LAB were isolated by differential centrifugation of samples of ruminal contents. The proportion of bacterial N in the incubation residues increased from 0.09 and 0.45 g bacterial N/g total N at 3 h of incubation to 0.37 and 0.85 g bacterial N/g total N at 48 h of incubation for early-cut and late-cut ryegrass, respectively. There were differences (P < 0.001) between uncorrected N degradability values and those corrected for microbial contamination with all of the methods. Apparent N degradability of the low-N, high-fiber forage (late-cut ryegrass) was 0.51, whereas the corrected values were 0.85, 0.84, and 0.77 for the LF, SAB, and LAB methods, respectively. With early-cut ryegrass and red clover, the differences between uncorrected and corrected values ranged between 6% and 13%, with small differences among the labeling methods. Generally, methods using labeled forage or labeled SAB and LAB provided similar corrected degradability values. The accuracy in estimating the extent of degradation of protein in the rumen from in situ disappearance curves is improved when values are corrected for microbial contamination of the bag residue.

Ammonia Assimilation in Zea mays L. Infected with a Vesicular-Arbuscular Mycorrhizal Fungus Glomus fasciculatum.

PubMed

Cliquet, J. B.; Stewart, G. R.

1993-03-01

To investigate nitrogen assimilation and translocation in Zea mays L. colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thax. sensu Gerd.), we measured key enzyme activities, 15N incorporation into free amino acids, and 15N translocation from roots to shoots. Glutamine synthetase and nitrate reductase activities were increased in both roots and shoots compared with control plants, and glutamate dehydrogenase activity increased in roots only. In the presence of [15N]ammonium, glutamine amide was the most heavily labeled product. More label was incorporated into amino acids in VAM plants. The kinetics of 15N labeling and effects of methionine sulfoximine on distribution of 15N-labeled products were entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. 15N translocation from roots to shoots through the xylem was higher in VAM plants compared with control plants. These results establish that, in maize, VAM fungi increase ammonium assimilation, glutamine production, and xylem nitrogen translocation. Unlike some ectomycorrhizal fungi, VAM fungi do not appear to alter the pathway of ammonium assimilation in roots of their hosts.

A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides

PubMed Central

Acedo, Montse; Fàbrega, Carme; Aviño, Anna; Goodman, Myron; Fagan, Patricia; Wemmer, David; Eritja, Ramon

1994-01-01

The use of the ammonia deprotection step to introduce 15N labels at specific exocyclic amino positions of adenine, cytosine, guanine or 2-aminopurine of oligodeoxynucleotides is described. PMID:8065910

Efficient assimilation of cyanobacterial nitrogen by water hyacinth.

PubMed

Qin, Hongjie; Zhang, Zhiyong; Liu, Minhui; Wang, Yan; Wen, Xuezheng; Yan, Shaohua; Zhang, Yingying; Liu, Haiqin

2017-10-01

A 15 N labeling technique was used to study nitrogen transfer from cyanobacterium Microcystis aeruginosa to water hyacinth. 15 N atom abundance in M. aeruginosa peaked (15.52%) after cultivation in 15 N-labeled medium for 3weeks. Over 87% of algal nitrogen was transferred into water hyacinth after the 4-week co-cultivation period. The nitrogen quickly super-accumulated in the water hyacinth roots, and the labeled nitrogen was re-distributed to different organs (i.e., roots, stalks, and leaves). This study provides a new strategy for further research on cyanobacterial bloom control, nitrogen migration, and nitrogen cycle in eutrophic waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

MASS LOSS AND NITROGEN DYNAMICS DURING THE DECOMPOSITION OF A N-LABELED N2-FIXING EPOPHYTIC LICHEN, LOBARIA OREGANA (TUCK.) MULL. ARG.

EPA Science Inventory

We studied mass loss and nitrogen dynamics during fall and spring initiated decomposition of an N2-fixing epiphytic lichen, Lobaria oregana (Tuck.) Mull. Arg. using 15N. We developed a method of labeling lichens with 15N that involved spraying lichen material with a nutrient sol...

[Dynamics of amino acid and protein metabolism in laying hens after the administration of 15N-labeled wheat protein. 11. Incorporation of 15N in the tissues and the amino acids of the muscles].

PubMed

Gruhn, K; Zander, R

1989-03-01

Over a period of 4 days 12 colostomized laying hens daily received 36 g 15N labelled wheat with 15N excess (15N') of 14.37 atom-% together with a conventional feed mixture for laying hens. The labelling of the lysine N in the wheat was 13.58 atom-%, that of histidine N 14.38 and that of arginine 15N' 13.63 atom-% 15N'. Three hens each were butchered 12, 36, 60 and 108 h after the last 15N' feeding. The first three hens did not receive any feed before being butchered. The following three hens each received the unlabelled feed ration for another 1, 2 or 4 days resp. after the main period until they were butchered. The total of skeleton muscles, the heart and the stomach muscle (without inner skin) of each hen were combined into one sample, cut thinly, drenched with fluid nitrogen and pulverized. N, 15N' and the basic and non-basic amino acids as well as their 15N' were determined in the individual samples. In contrast to the organs, the proteins in the muscle tissue have a long half life so that a slight decrease of atom-% 15N' in the muscles could only be detected after 108 h. The 14N and 15N' quota of the non-basic amino acids in the total nitrogen of the muscles is 50%. The 14N quota of the basic amino acids is 30% and the 15N' quota only 22.5% in the total muscle N. The heavy nitrogen of the free lysine in the TCA soluble N fraction is hardly detectable 36 h and 60 h after the last 15N' supply and not at all after 108 h. In contrast to this, the other two free basic amino acids remain significantly higher labelled in dependence on the last butchering time.

Application of isotopic techniques to investigate the impact of insect herbivory on C and N cycling in a grassland system - a mesocosm study

NASA Astrophysics Data System (ADS)

Potthast, Karin; Meyer, Stefanie; Gleixner, Gerd; Crecelius, Anna; Schubert, Ulrich; Michalzik, Beate

2017-04-01

Ecosystem disturbances like insect pests induce time and space limited process heterogeneity that allow to quantify changes in biogeochemical reaction rates. Insect pests are known to impact element and organic matter (OM) cycling in ecosystems by defoliation and deposition of fecal material. To study the effects of such trophic interactions on OM and nutrient cycling in a grassland system under herbivore attack, a laboratory mesocosm experiment with grass (Dactylis glomerata) and grasshoppers (Chorthippus dorsatus) was conducted. In 12 mesocosms (50 cm in diameter, 100 cm in height) D. glomerata was sown in pasture topsoil (0-12 cm of a Calcaric Cambisol (Siltic), Hainich region, Germany) and left to grow for one year under constant climatic conditions (15°C) to establish a well-developed root system. In 2015, the mesocosm was labeled over 5 days using 13CO2-gas and 15N labeled feces (δ15N: 58‰) in order to trace the fate of C and N in above- and belowground plant organs (root, leave), insects, feces, soil, and soil solution. In three replicates, the following treatments were conducted: control, 13CO2-labelling, 13CO2-labelling+20 grasshoppers, and 13CO2-labelling+20 grasshoppers +15N-labeled feces (+9.2 µg N*cm-2). During incubation, the mesocosms were irrigated (13 mm) and throughfall and soil solutions were sampled. After incubation, solutions, cold water extracts as well as microbial biomass (chloroform-fumigation) of two soil depths (0-4, 4-12 cm) were analyzed for DOC, δ13DOC, and dissolved N. Furthermore, TOC, δ13C, TN and δ15N values of all collected compartments were determined. In general, 13CO2-pulse labelling showed that after 5 days of incubation not only grasshopper feces but also leachates of feces were significantly enriched in 13C. Based on δ13C-values, herbivory induced a stronger 13C-enrichment in roots while shoots were less enriched. The input of 13DOC indicates a fast cycling of leaf-C via grasshopper and feces to the soil solution, soil microbes and grass roots. This was further confirmed by a 80 % mass loss and by a reduced N amount (-91%) of labeled feces. This may indicate a rapid release of N via leaching, and root-uptake (-0.82±0.28‰) compared to treatments without 15N (-1.54±0.12‰). 15N in grass leaves was not found to be enriched, however, significantly higher δ15N values were found in freshly excreted feces (0.62±0.4‰) compared to those of mesocosms without 15N addition (-0.14 ±0.27‰). Hence, we hypothesize that part of the labeled N was also rapidly assimilated within plant and microbial biomass, taken up by grasshoppers, and returned via feces. The N amount in soil solution and cold water extracts did not increase due to herbivory supporting the assumption of a rapid plant uptake of released N. The low N concentrations of the mineral soil (0.14 %) and in soil solutions (1-2.3 mg L-1) point to very low N availability for the grass. We conclude that on the short-time scale in N-limited pasture systems heavy insect herbivory would not result in significant N leaching from the ecosystem.

Biosynthesis and characterization of ¹⁵N₆-labeled phomopsin A, a lupin associated mycotoxin produced by Diaporthe toxica.

PubMed

Schloß, Svenja; Wedell, Ines; Koch, Matthias; Rohn, Sascha; Maul, Ronald

2015-06-15

The hepatotoxin phomopsin A (PHO-A), a secondary metabolite mainly produced by the fungus Diaporthe toxica, occurs predominantly on sweet lupins. Along with the growing interest in sweet lupins for food and feed commodities, concerns have been raised about fungal infestations, and consequently, about the determination of PHO-A. High performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) represents the most suitable analytical technique for sensitive and selective detection of mycotoxins including PHO-A. However, isotopic labeled substances are needed as internal standards for a reliable and convenient quantification. As no isotope standard for PHO-A is currently available, a biosynthesis of fully (15)N6-labeled PHO-A was established by cultivation of D. toxica on defined media containing Na(15)NO3 and (15)N-labeled yeast extract as the only nitrogen sources. The identity of (15)N6-PHO-A was confirmed by high resolution mass spectrometry. The new (15)N6-labeled standard will facilitate the method development for PHO-A including a more accurate quantification by LC-MS/MS. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effective rotational correlation times of proteins from NMR relaxation interference

NASA Astrophysics Data System (ADS)

Lee, Donghan; Hilty, Christian; Wider, Gerhard; Wüthrich, Kurt

2006-01-01

Knowledge of the effective rotational correlation times, τc, for the modulation of anisotropic spin-spin interactions in macromolecules subject to Brownian motion in solution is of key interest for the practice of NMR spectroscopy in structural biology. The value of τc enables an estimate of the NMR spin relaxation rates, and indicates possible aggregation of the macromolecular species. This paper reports a novel NMR pulse scheme, [ 15N, 1H]-TRACT, which is based on transverse relaxation-optimized spectroscopy and permits to determine τc for 15N- 1H bonds without interference from dipole-dipole coupling of the amide proton with remote protons. [ 15N, 1H]-TRACT is highly efficient since only a series of one-dimensional NMR spectra need to be recorded. Its use is suggested for a quick estimate of the rotational correlation time, to monitor sample quality and to determine optimal parameters for complex multidimensional NMR experiments. Practical applications are illustrated with the 110 kDa 7,8-dihydroneopterin aldolase from Staphylococcus aureus, the uniformly 15N-labeled Escherichia coli outer membrane protein X (OmpX) in 60 kDa mixed OmpX/DHPC micelles with approximately 90 molecules of unlabeled 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC), and the 16 kDa pheromone-binding protein from Bombyx mori, which cover a wide range of correlation times.

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

Selective excitation for spectral editing and assignment in separated local field experiments of oriented membrane proteins

NASA Astrophysics Data System (ADS)

Koroloff, Sophie N.; Nevzorov, Alexander A.

2017-01-01

Spectroscopic assignment of NMR spectra for oriented uniformly labeled membrane proteins embedded in their native-like bilayer environment is essential for their structure determination. However, sequence-specific assignment in oriented-sample (OS) NMR is often complicated by insufficient resolution and spectral crowding. Therefore, the assignment process is usually done by a laborious and expensive "shotgun" method involving multiple selective labeling of amino acid residues. Presented here is a strategy to overcome poor spectral resolution in crowded regions of 2D spectra by selecting resolved "seed" residues via soft Gaussian pulses inserted into spin-exchange separated local-field experiments. The Gaussian pulse places the selected polarization along the z-axis while dephasing the other signals before the evolution of the 1H-15N dipolar couplings. The transfer of magnetization is accomplished via mismatched Hartmann-Hahn conditions to the nearest-neighbor peaks via the proton bath. By optimizing the length and amplitude of the Gaussian pulse, one can also achieve a phase inversion of the closest peaks, thus providing an additional phase contrast. From the superposition of the selective spin-exchanged SAMPI4 onto the fully excited SAMPI4 spectrum, the 15N sites that are directly adjacent to the selectively excited residues can be easily identified, thereby providing a straightforward method for initiating the assignment process in oriented membrane proteins.

Pathways of assimilation and transfer of fixed nitrogen in coralloid roots of cycad-Nostoc symbioses.

PubMed

Pate, J S; Lindblad, P; Atkins, C A

1988-12-01

Freshly detached coralloid roots of several cycad species were found to bleed spontaneously from xylem, permitting identification of products of nitrogen transfer from symbiotic organ to host. Structural features relevant to the export of fixed N were described for Macrozamia riedlei (Fisch. ex Gaud.) Gardn. the principal species studied. Citrulline (Cit), glutamine (Gln) and glutamic acid (Glu), the latter usually in a lesser amount, were the principal translocated solutes in Macrozamia (5 spp.), Encephalartos (4 spp.) and Lepidozamia (1 sp.), while Gln and a smaller amount of Glu, but no Cit were present in xylem sap of Bowenia (1 sp.),and Cycas (2 spp.). Time-course studies of (15)N enrichment of the different tissue zones and the xylem sap of (15)N2-pulse-fed coralloid roots of M. riedlei showed earlier (15)N incorporation into Gln than into Cit, and a subsequent net decline in the (15)N of Gln of the coralloid-root tissues, whereas Cit labeling continued to increase in inner cortex and stele and in the xylem sap. Hydrolysis of the (15)N-labeled Cit and Gln consistently demonstrated much more intense labeling of the respective carbamyl and amide groups than of the other N-atoms. Coralloid roots of M. riedlei pulse-fed (14)CO2 in darkness showed (14)C labeling of aspartic acid (Asp) and Cit in all tissue zones and of Cit of xylem bleeding sap. Lateral roots and uninfected apogeotropic roots of M. riedlei and M. moorei also incorporated (14)CO2 into Cit. The (14)C of Cit was restricted to the carbamyl-C. Comparable (15)N2 and CO2-feeding studies on corallid roots of Cycas revoluta showed Gln to be the dominant product of N2 fixation, with Asp and alanine as other major (14)C-labeled amino compounds, but a total absence of Cit in labeled or unlabeled form.

Variable Nitrogen Fixation in Wild Populus

PubMed Central

Doty, Sharon L.; Sher, Andrew W.; Fleck, Neil D.; Khorasani, Mahsa; Bumgarner, Roger E.; Khan, Zareen; Ko, Andrew W. K.; Kim, Soo-Hyung; DeLuca, Thomas H.

2016-01-01

The microbiome of plants is diverse, and like that of animals, is important for overall health and nutrient acquisition. In legumes and actinorhizal plants, a portion of essential nitrogen (N) is obtained through symbiosis with nodule-inhabiting, N2-fixing microorganisms. However, a variety of non-nodulating plant species can also thrive in natural, low-N settings. Some of these species may rely on endophytes, microorganisms that live within plants, to fix N2 gas into usable forms. Here we report the first direct evidence of N2 fixation in the early successional wild tree, Populus trichocarpa, a non-leguminous tree, from its native riparian habitat. In order to measure N2 fixation, surface-sterilized cuttings of wild poplar were assayed using both 15N2 incorporation and the commonly used acetylene reduction assay. The 15N label was incorporated at high levels in a subset of cuttings, suggesting a high level of N-fixation. Similarly, acetylene was reduced to ethylene in some samples. The microbiota of the cuttings was highly variable, both in numbers of cultured bacteria and in genetic diversity. Our results indicated that associative N2-fixation occurred within wild poplar and that a non-uniformity in the distribution of endophytic bacteria may explain the variability in N-fixation activity. These results point to the need for molecular studies to decipher the required microbial consortia and conditions for effective endophytic N2-fixation in trees. PMID:27196608

RAPID AND AUTOMATED PROCESSING OF MALDI-FTICR/MS DATA FOR N-METABOLIC LABELING IN A SHOTGUN PROTEOMICS ANALYSIS.

PubMed

Jing, Li; Amster, I Jonathan

2009-10-15

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

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

Treesearch

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

2013-01-01

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

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

NASA Technical Reports Server (NTRS)

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

1991-01-01

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

Simplified Synthesis of Isotopically Labeled 5,5-Dimethyl-pyrroline N-Oxide

PubMed Central

Leinisch, Fabian; Jiang, JinJie; Deterding, Leesa J.; Mason, Ronald P.

2011-01-01

5,5-Dimethylpyrroline N-oxide (15N) and 5,5-di(trideuteromethyl)pyrroline N-oxide were synthesized from the respective isotopically labeled 2-nitropropane analogs obtained from the reaction of sodium nitrate with 2-halopropanes. This facile, straightforward process allows synthesizing isotopically labeled DMPO analogs in a 4-step reaction without special equipment. PMID:21986521

Heteronuclear proton assisted recoupling

NASA Astrophysics Data System (ADS)

De Paëpe, Gaël; Lewandowski, Józef R.; Loquet, Antoine; Eddy, Matt; Megy, Simon; Böckmann, Anja; Griffin, Robert G.

2011-03-01

We describe a theoretical framework for understanding the heteronuclear version of the third spin assisted recoupling polarization transfer mechanism and demonstrate its potential for detecting long-distance intramolecular and intermolecular 15N-13C contacts in biomolecular systems. The pulse sequence, proton assisted insensitive nuclei cross polarization (PAIN-CP) relies on a cross term between 1H-15N and 1H-13C dipolar couplings to mediate zero- and/or double-quantum 15N-13C recoupling. In particular, using average Hamiltonian theory we derive effective Hamiltonians for PAIN-CP and show that the transfer is mediated by trilinear terms of the form N±C∓Hz (ZQ) or N±C±Hz (DQ) depending on the rf field strengths employed. We use analytical and numerical simulations to explain the structure of the PAIN-CP optimization maps and to delineate the appropriate matching conditions. We also detail the dependence of the PAIN-CP polarization transfer with respect to local molecular geometry and explain the observed reduction in dipolar truncation. In addition, we demonstrate the utility of PAIN-CP in structural studies with 15N-13C spectra of two uniformly 13C,15N labeled model microcrystalline proteins—GB1, a 56 amino acid peptide, and Crh, a 85 amino acid domain swapped dimer (MW = 2 × 10.4 kDa). The spectra acquired at high magic angle spinning frequencies (ωr/2π > 20 kHz) and magnetic fields (ω0H/2π = 700-900 MHz) using moderate rf fields, yield multiple long-distance intramonomer and intermonomer 15N-13C contacts. We use these distance restraints, in combination with the available x-ray structure as a homology model, to perform a calculation of the monomer subunit of the Crh protein.

In situ 15N labeling experiment reveals different long-term responses to ammonium and nitrate inputs in N-saturated subtropical forest

NASA Astrophysics Data System (ADS)

Liu, Wenjing; Yu, Longfei; Zhang, Ting; Kang, Ronghua; Zhu, Jing; Mulder, Jan; Huang, Yongmei; Duan, Lei

2017-09-01

Chronically elevated deposition of reactive nitrogen (N), as ammonium (NH4+) and nitrate (NO3-), in subtropical forests with monsoonal climate has caused widespread N leaching in southern China. So far, little is known about the effect of further increases in N input and changes in the relative proportion of NH4+ and NO3- on turnover rate and fate of atmogenic N. Here we report a 15N tracer experiment in Tieshanping (TSP) forest, SW China, conducted as part of a long-term N fertilization experiment, using NH4NO3 and NaNO3, where effects of a doubling of monthly N inputs were compared. In June 2012, the regular N fertilizers were replaced by their 15N-labeled forms, viz., 15NH4NO3 and Na15NO3, as a single-dose addition. Mass balances of N for the initial 1.5 years following label addition showed that for both treatments, 70% to 80% of the annual N input was leached as NO3-, both at ambient and at double N input rates. This confirms the earlier reported extreme case of N saturation at TSP. The 15N, added as Na15NO3, showed recoveries of about 74% in soil leachates, indicating that NO3- input at TSP is subject to a rapid and nearly quantitative loss through direct leaching as a mobile anion. By contrast, recoveries of 15N in soil leachates of only 33% were found if added as 15NH4NO3. Much of the 15N was immobilized in the soil and to a lesser extent in the vegetation. Thus, immobilization of fresh N input is significantly greater if added as NH4+, than as NO3-.

15N-labeled glycine synthesis.

PubMed

Tavares, Claudinéia R O; Bendassolli, José A; Coelho, Fernando; Sant'ana Filho, Carlos R; Prestes, Clelber V

2006-09-01

This work describes a method for 15N-isotope-labeled glycine synthesis, as well as details about a recovery line for nitrogen residues. To that effect, amination of alpha-haloacids was performed, using carboxylic chloroacetic acid and labeled aqueous ammonia (15NH3). Special care was taken to avoid possible 15NH3 losses, since its production cost is high. In that respect, although the purchase cost of the 13N-labeled compound (radioactive) is lower, the stable tracer produced constitutes an important tool for N cycling studies in living organisms, also minimizing labor and environmental hazards, as well as time limitation problems in field studies. The tests were carried out with three replications, and variable 15NH3aq volumes in the reaction were used (50, 100, and 150 mL), in order to calibrate the best operational condition; glycine masses obtained were 1.7, 2, and 3.2 g, respectively. With the development of a system for 15NH3 recovery, it was possible to recover 71, 83, and 87% of the ammonia initially used in the synthesis. With the required adaptations, the same system was used to recover methanol, and 75% of the methanol initially used in the amino acid purification process were recovered.

Initial association of fresh microbial products to soil particles: a joint density fractionation and NanoSIMS study

NASA Astrophysics Data System (ADS)

Hatton, Pierre-Joseph; Remusat, Laurent; Brewer, Elizabeth; Derrien, Delphine

2014-05-01

While soil microorganisms are increasingly seen as shaping stable soil organic matter (OM) formation, the mechanisms controlling the attachment of microbial metabolites to soil particles are not fully understood yet. We investigate the spatial distribution of freshly produced microbial products among density-isolated fractions of soil using stable C and N isotopes and Nano-scale secondary ion mass spectrometry (NanoSIMS). A surface forest soil was amended with uniformly 13C/15N labeled glycine and incubated for 8 hours in gamma-irradiated and non-sterile soils. Sequential density fractionation was then performed to isolate various classes of aggregates and of single mineral particles. Eight hours after the labeled glycine addition, 7 % of the 13C and 15N was tightly bound to soil assemblages. Comparison of sterile and non-sterile treatments revealed that microbial activity was almost completely responsible for this rapid association (>85 %). The distributions of glycine-derived 13C and 15N, considered as markers of new microbial products, were mapped on particles of the non-sterile treatment using NanoSIMS. New microbial products were heterogeneously distributed and spatially decoupled at the surface of on soil particles. 13C microbial products were scarce and presumably within or in the vicinity of microbial cells. In contrast, 15N microbial products seemed evenly spread at the surface of soil particles, likely as soluble exoenzymes diffusing away from their parent cell. Macroscopic measurements among density fractions suggested that the diffusion of such 15N microbial products was spatially limited yet, because of pore space architecture. NanoSIMS images further allowed gaining insight into the attachment of the new microbial products on particle surfaces already covered by OM, in a multilayer fashion. Using an internal calibration method to determine C/N ratios of NanoSIMS images, we showed the preferential attachment of soluble microbial N-metabolites to N-rich mineral-attached OM (C/N ratios mostly < 16). Exceptions were found in dense particles, supposed to contained aluminium and iron (hydr)oxides, with the microbial N-metabolites apparently preferentially attached to C-rich mineral-attached OM (C/N ratios > 80). This work provided visual evidences that the attachment of new microbial products to the soil matrix is mediated by distinct processes for N-rich and C-rich metabolites. It also demonstrated that the pore space architecture has impact on the formation of stable OM by limiting the diffusion of soluble microbial metabolites and their access to reactive and stabilising surfaces.

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

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

We report on the contamination of commercial 15-nitrogen (15N) N2 gas stocks with 15N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. 15N2 gas is used to estimate N2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled 15N2 into organic matter. However, the microbial assimilation of bioavailable 15N-labeled N2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N2 fixation rates. 15N2 gas procured from three major suppliers was analyzed for the presence of these 15N-contaminants. Substantial concentrations of 15N-contaminants were detected in four Sigma-Aldrich 15N2 lecture bottles from two discrete batch syntheses. Per mole of 15N2 gas, 34 to 1900 µmoles of 15N-ammonium, 1.8 to 420 µmoles of 15N-nitrate/nitrite, and ≥21 µmoles of 15N-nitrous oxide were detected. One 15N2 lecture bottle from Campro Scientific contained ≥11 µmoles of 15N-nitrous oxide per mole of 15N2 gas, and no detected 15N-nitrate/nitrite at the given experimental 15N2 tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles 15N-nitrous oxide per mole 15N2, and trace concentrations of 15N-ammonium and 15N-nitrate/nitrite. 15N2 gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the 15N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N2 fixation assays suggests that the degree of detected 15N-ammonium contamination could yield inferred N2 fixation rates ranging from undetectable, <0.01 nmoles N L−1 d−1, to 530 nmoles N L−1 d−1, contingent on experimental conditions. These rates are comparable to, or greater than, N2 fixation rates commonly detected in field assays. These results indicate that past reports of N2 fixation should be interpreted with caution, and demonstrate that the purity of commercial 15N2 gas must be ensured prior to use in future N2 fixation rate determinations. PMID:25329300

Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

PubMed Central

Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

2010-01-01

We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006

Post-harvest N2O emissions were not affected by various types of oilseed straw incorporated into soil

NASA Astrophysics Data System (ADS)

Köbke, Sarah; Senbayram, Mehmet; Hegewald, Hannes; Christen, Olaf; Dittert, Klaus

2015-04-01

Oilseed rape post-harvest N2O emissions are seen highly critical as so far they are considered as one of the most crucial drawbacks in climate-saving bioenergy production systems. N2O emissions may substantially counterbalance the intended savings in CO2 emissions. Carbon-rich crop residues in conjunction with residual soil nitrate are seen as a key driver since they may serve as energy source for denitrification and, they may alter soil-borne N2O emissions. As oilseed rape straw is known to have high N/C ratio compared to other crop residues, its soil incorporation may specifically trigger post-harvest N2O emissions. Therefore, the aim of the present study was to determine post-harvest N2O emissions in soils amended with various types of oilseed rape straw (with different N/C ratio) and barley straw in field and incubation experiments. In the incubation experiment, oilseed rape or 15N labelled barley straw were mixed with soil at a rate of 1.3 t DM ha-1 and studied for 43 days. Treatments consisted of non-treated control soil (CK), 15N labelled barley straw (BST), oilseed rape straw (RST), 15N labelled barley straw + N (BST+N), or oilseed rape straw + N (RST+N). N fertilizer was applied to the soil surface as ammonium-nitrate at a rate of 100 kg N ha-1 and soil moisture was adjusted to 80% water-holding capacity. In the field experiment, during the vegetation period 15N labelled fertilizer (15NH415NO3) was used to generate 15N labelled oilseed rape straw (up to 5 at%). Here, the three fertilizer treatments consisted of 5 kg N ha-1 (RST-5), 150 kg N ha-1 (RST-150) and 180 kg N ha-1 (RST-180). Post-harvest N2O emissions were determined during the period of August 2013 to February 2014 by using static flux chambers. In the incubation trial, cumulative N2O emissions were 5, 29, 40 g N2O-N ha-1 148 days-1 in non-fertilized control, BST and RST treatments, respectively. Here, emissions were slightly higher in RST than BST (p

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method.

PubMed

Takeda, Mitsuhiro; Chang, Chung-ke; Ikeya, Teppei; Güntert, Peter; Chang, Yuan-hsiang; Hsu, Yen-lan; Huang, Tai-huang; Kainosho, Masatsune

2008-07-18

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform (13)C and (15)N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the beta-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.

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

NASA Astrophysics Data System (ADS)

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

2014-05-01

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

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

PubMed

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

2015-02-01

Although ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is mostly known as a key enzyme involved in CO2 assimilation during the Calvin cycle, comparatively little is known about its role as a pool of nitrogen storage in leaves. For this purpose, we developed a protocol to purify Rubisco that enables later analysis of its (15) N isotope composition (δ(15) N) at the natural abundance and (15) N-labeled plants. In order to test the utility of this protocol, durum wheat (Triticum durum var. Sula) exposed to an elevated CO2 concentration (700 vs 400 µmol mol(-1) ) was labeled with K(15) NO3 (enriched at 2 atom %) during the ear development period. The developed protocol proves to be selective, simple, cost effective and reproducible. The study reveals that (15) N labeling was different in total organic matter, total soluble protein and the Rubisco fraction. The obtained data suggest that photosynthetic acclimation in wheat is caused by Rubisco depletion. This depletion may be linked to preferential nitrogen remobilization from Rubisco toward grain filling. © 2014 Scandinavian Plant Physiology Society.

Assimilation and partitioning of prey nitrogen within two anthozoans and their endosymbiotic zooxanthellae

USGS Publications Warehouse

Piniak, G.A.; Lipschultz, F.; McClelland, J.

2003-01-01

The movement of nitrogen from zooplankton prey into the temperate scleractinian coral Oculina arbuscula and the anemone Aiptasia pallida was measured using 15N-labeled brine shrimp. The efficiency with which prey nitrogen was incorporated into cnidarian tissues was species-specific. O. arbuscula with a full complement of zooxanthellae had an assimilation efficiency of nearly 100%, compared to only 46% for corals containing few zooxanthellae. In A. pallida, symbiont density had no effect, and nitrogen assimilation was 23 to 29%. In both species, the host retained the bulk of the ingested label. Complete digestion was rapid (<4 h), as was the partitioning of the label between host amino acids and macromolecules. The label was primarily in the low-molecular weight-amino acid pool in O. arbuscula, where it remained for 30 h. A maximum of ca. 20% of the 15N appeared in the zooxanthellae, where it was rapidly converted into macromolecules. Individual amino acids in A. pallida tissues were highly labeled with 15N within 4 h and showed no subsequent enrichment with time; however, zooxanthellae amino acids became increasingly enriched over 30 h. Differences in 15N enrichment among amino acids were consistent with known synthesis and transformation pathways, but it was not possible to discriminate between host feeding and de novo synthesis.

15N NATURAL ABUNDANCE AND 15N LABELLING STUDIES IN FOREST ECOSYSTEMS

EPA Science Inventory

The relative amounts of the two stable isotopes of Nitrogen (N), 15N, and N, vary predictably in soils and plant tissues of forests and other non-cultivated ecosystems. light fractionations, or discriminations against the heavier N isotope, that can occur as N cycles through vege...

Spectral density mapping at multiple magnetic fields suitable for 13C NMR relaxation studies

NASA Astrophysics Data System (ADS)

Kadeřávek, Pavel; Zapletal, Vojtěch; Fiala, Radovan; Srb, Pavel; Padrta, Petr; Přecechtělová, Jana Pavlíková; Šoltésová, Mária; Kowalewski, Jozef; Widmalm, Göran; Chmelík, Josef; Sklenář, Vladimír; Žídek, Lukáš

2016-05-01

Standard spectral density mapping protocols, well suited for the analysis of 15N relaxation rates, introduce significant systematic errors when applied to 13C relaxation data, especially if the dynamics is dominated by motions with short correlation times (small molecules, dynamic residues of macromolecules). A possibility to improve the accuracy by employing cross-correlated relaxation rates and on measurements taken at several magnetic fields has been examined. A suite of protocols for analyzing such data has been developed and their performance tested. Applicability of the proposed protocols is documented in two case studies, spectral density mapping of a uniformly labeled RNA hairpin and of a selectively labeled disaccharide exhibiting highly anisotropic tumbling. Combination of auto- and cross-correlated relaxation data acquired at three magnetic fields was applied in the former case in order to separate effects of fast motions and conformational or chemical exchange. An approach using auto-correlated relaxation rates acquired at five magnetic fields, applicable to anisotropically moving molecules, was used in the latter case. The results were compared with a more advanced analysis of data obtained by interpolation of auto-correlated relaxation rates measured at seven magnetic fields, and with the spectral density mapping of cross-correlated relaxation rates. The results showed that sufficiently accurate values of auto- and cross-correlated spectral density functions at zero and 13C frequencies can be obtained from data acquired at three magnetic fields for uniformly 13C -labeled molecules with a moderate anisotropy of the rotational diffusion tensor. Analysis of auto-correlated relaxation rates at five magnetic fields represents an alternative for molecules undergoing highly anisotropic motions.

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

PubMed

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

2017-01-01

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

Quantifying the effect of plant growth on litter decomposition using a novel, triple-isotope label approach

NASA Astrophysics Data System (ADS)

Ernakovich, J. G.; Baldock, J.; Carter, T.; Davis, R. A.; Kalbitz, K.; Sanderman, J.; Farrell, M.

2017-12-01

Microbial degradation of plant detritus is now accepted as a major stabilizing process of organic matter in soils. Most of our understanding of the dynamics of decomposition come from laboratory litter decay studies in the absence of plants, despite the fact that litter decays in the presence of plants in many native and managed systems. There is growing evidence that living plants significantly impact the degradation and stabilization of litter carbon (C) due to changes in the chemical and physical nature of soils in the rhizosphere. For example, mechanistic studies have observed stimulatory effects of root exudates on litter decomposition, and greenhouse studies have shown that living plants accelerate detrital decay. Despite this, we lack a quantitative understanding of the contribution of living plants to litter decomposition and how interactions of these two sources of C build soil organic matter (SOM). We used a novel triple-isotope approach to determine the effect of living plants on litter decomposition and C cycling. In the first stage of the experiment, we grew a temperate grass commonly used for forage, Poa labillardieri, in a continuously-labelled atmosphere of 14CO2 fertilized with K15NO3, such that the grass biomass was uniformly labelled with 14C and 15N. In the second stage, we constructed litter decomposition mescososms with and without a living plant to test for the effect of a growing plant on litter decomposition. The 14C/15N litter was decomposed in a sandy clay loam while a temperate forage grass, Lolium perenne, grew in an atmosphere of enriched 13CO2. The fate of the litter-14C/15N and plant-13C was traced into soil mineral fractions and dissolved organic matter (DOM) over the course of nine weeks using four destructive harvests of the mesocosms. Our preliminary results suggest that living plants play a major role in the degradation of plant litter, as litter decomposition was greater, both in rate and absolute amount, for soil mesocosms with a growing plant. Our observations during the decomposition experiment suggests that plant roots physically disrupted litter to increase decomposition. Isotopic analyses are currently underway, and transformations of litter-14C will be presented. Refining our understanding of in situ litter decay will add to our growing knowledge of the C cycle.

Greater carbon allocation to mycorrhizal fungi reduces tree nitrogen uptake in a boreal forest.

PubMed

Hasselquist, Niles J; Metcalfe, Daniel B; Inselsbacher, Erich; Stangl, Zsofia; Oren, Ram; Näsholm, Torgny; Högberg, Peter

2016-04-01

The central role that ectomycorrhizal (EM) symbioses play in the structure and function of boreal forests pivots around the common assumption that carbon (C) and nitrogen (N) are exchanged at rates favorable for plant growth. However, this may not always be the case. It has been hypothesized that the benefits mycorrhizal fungi convey to their host plants strongly depends upon the availability of C and N, both of which are rapidly changing as a result of intensified human land use and climate change. Using large-scale shading and N addition treatments, we assessed the independent and interactive effects of changes in C and N supply on the transfer of N in intact EM associations with -15 yr. old Scots pine trees. To assess the dynamics of N transfer in EM symbioses, we added trace amounts of highly enriched 5NO3(-) label to the EM-dominated mor-layer and followed the fate of the 15N label in tree foliage, fungal chitin on EM root tips, and EM sporocarps. Despite no change in leaf biomass, shading resulted in reduced tree C uptake, ca. 40% lower fungal biomass on EM root tips, and greater 15N label in tree foliage compared to unshaded control plots, where more 15N label was found in fungal biomass on EM colonized root tips. Short-term addition of N shifted the incorporation of 15N label from EM fungi to tree foliage, despite no significant changes in below-ground tree C allocation to EM fungi. Contrary to the common assumption that C and N are exchanged at rates favorable for plant growth, our results show for the first time that under N-limited conditions greater C allocation to EM fungi in the field results in reduced, not increased, N transfer to host trees. Moreover, given the ubiquitous nature of mycorrhizal symbioses, our results stress the need to incorporate mycorrhizal dynamics into process-based ecosystem models to better predict forest C and N cycles in light of global climate change.

Artifact suppression in electron paramagnetic resonance imaging of 14N- and 15N-labeled nitroxyl radicals with asymmetric absorption spectra

NASA Astrophysics Data System (ADS)

Takahashi, Wataru; Miyake, Yusuke; Hirata, Hiroshi

2014-10-01

This article describes an improved method for suppressing image artifacts in the visualization of 14N- and 15N-labeled nitroxyl radicals in a single image scan using electron paramagnetic resonance (EPR). The purpose of this work was to solve the problem of asymmetric EPR absorption spectra in spectral processing. A hybrid function of Gaussian and Lorentzian lineshapes was used to perform spectral line-fitting to successfully separate the two kinds of nitroxyl radicals. This approach can process the asymmetric EPR absorption spectra of the nitroxyl radicals being measured, and can suppress image artifacts due to spectral asymmetry. With this improved visualization method and a 750-MHz continuous-wave EPR imager, a temporal change in the distributions of a two-phase paraffin oil and water/glycerin solution system was visualized using lipophilic and hydrophilic nitroxyl radicals, i.e., 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy (16-DOXYL stearic acid) and 4-hydroxyl-2,2,6,6-tetramethylpiperidine-d17-1-15N-1-oxyl (TEMPOL-d17-15N). The results of the two-phase separation experiment verified that reasonable artifact suppression could be achieved by the present method that deals with asymmetric absorption spectra in the EPR imaging of 14N- and 15N-labeled nitroxyl radicals.

A Cluster Analytic Study of Clinical Orientations among Chemical Dependency Counselors.

ERIC Educational Resources Information Center

Thombs, Dennis L.; Osborn, Cynthia J.

2001-01-01

Three distinct clinical orientations were identified in a sample of chemical dependency counselors (N=406). Based on cluster analysis, the largest group, identified and labeled as "uniform counselors," endorsed a simple, moral-disease model with little interest in psychosocial interventions. (Contains 50 references and 4 tables.) (GCP)

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

PubMed

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

2017-08-18

The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13 C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15 N-labelled amino acids could be obtained.

Staphylococcus aureus Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy

PubMed Central

Kim, Sung Joon; Singh, Manmilan; Preobrazhenskaya, Maria; Schaefer, Jacob

2013-01-01

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with D-[1-13C]alanine and L-[15N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between 13C and 15N of labeled amino acids incorporated in the peptidoglycan, and from those labels to 19F of a glycopeptide drug specifically bound to the peptidoglycan. The observed 13C-15N average distance of 4.1 to 4.4 Å between D- and L-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus. PMID:23617832

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

PubMed Central

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

2011-01-01

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

Ability of L-canavanine to support nitrogen metabolism in the jack bean, Canavalia ensiformis (L. ) DC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenthal, G.A.; Berge, M.A.; Ozinskas, A.J.

The ability of L-canavanine, a nonprotein amino acid of certain leguminous plants, to support the nitrogen metabolism of jack bean, Canavalia ensiformis (Leguminosae), was assessed by administration of L-(guanidino-N{sup 3}-{sup 15}N)arginine, L-(guanidinooxy-N{sup 3}-{sup 15}N)canavanine, or L-(guanidinooxy-N{sup 1}-{sup 15}N)canavanine into the cotyledons of 9-day-old plants. A strikingly similar pattern of {sup 15}N assimilation into de novo synthesized amino and imino acids resulted from feeding L-(guanidino-N{sup 3}-{sup 15}N)arginine and L-(guanidinooxy-N{sup 3}-{sup 15}N)canavanine. Glutamic acid plus glutamine and alanine were the most heavily labeled of the detected compounds. Some transfer of {sup 15}N from L-(gluanidino-N{sup 3}-{sup 15}N)arginine to canavanine was noted. This maymore » occur by a transamidination reaction between L-canaline and L-arginine. L-(guanidinooxy-N{sup 1}-{sup 15}N)Canavanine also supported amino and imino acid biosynthesis in this plant, but much more alanine and less glutamic acid and glutamine were labeled. These experiments provide substantive experimental evidence for the long-reputed hypothesis that canavanine functions as a nitrogen-storing metabolite.« less

77 FR 70885 - Uniform Compliance Date for Food Labeling Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-28

... in the following way: Federal eRulemaking Portal: http://www.regulations.gov . Follow the... periodically have announced uniform compliance dates for new food labeling requirements (see, e.g., the Federal... development of new labeling materials. This policy serves consumers' interests as well because the cost of...

Resonance assignment of disordered protein with repetitive and overlapping sequence using combinatorial approach reveals initial structural propensities and local restrictions in the denatured state.

PubMed

Malik, Nikita; Kumar, Ashutosh

2016-09-01

NMR resonance assignment of intrinsically disordered proteins poses a challenge because of the limited dispersion of amide proton chemical shifts. This becomes even more complex with the increase in the size of the system. Residue specific selective labeling/unlabeling experiments have been used to resolve the overlap, but require multiple sample preparations. Here, we demonstrate an assignment strategy requiring only a single sample of uniformly labeled (13)C,(15)N-protein. We have used a combinatorial approach, involving 3D-HNN, CC(CO)NH and 2D-MUSIC, which allowed us to assign a denatured centromeric protein Cse4 of 229 residues. Further, we show that even the less sensitive experiments, when used in an efficient manner can lead to the complete assignment of a complex system without the use of specialized probes in a relatively short time frame. The assignment of the amino acids discloses the presence of local structural propensities even in the denatured state accompanied by restricted motion in certain regions that provides insights into the early folding events of the protein.

Cluster Roots of Leucadendron laureolum (Proteaceae) and Lupinus albus (Fabaceae) Take Up Glycine Intact: An Adaptive Strategy to Low Mineral Nitrogen in Soils?

PubMed Central

HAWKINS, HEIDI-JAYNE; WOLF, GABRIELLE; STOCK, WILLIAM DAVID

2005-01-01

• Background and Aims South African soils are not only low in phosphorus (P) but most nitrogen (N) is in organic form, and soil amino acid concentrations can reach 2·6 g kg−1 soil. The Proteaceae (a main component of the South African Fynbos vegetation) and some Fabaceae produce cluster roots in response to low soil phosphorus. The ability of these roots to acquire the amino acid glycine (Gly) was assessed. • Methods Uptake of organic N as 13C–15N-Gly was determined in cluster roots and non-cluster roots of Leucadendron laureolum (Proteaceae) and Lupinus albus (Fabaceae) in hydroponic culture, taking account of respiratory loss of 13CO2. • Key Results Both plant species acquired doubly labelled (intact) Gly, and respiratory losses of 13CO2 were small. Lupin (but not leucadendron) acquired more intact Gly when cluster roots were supplied with 13C–15N-Gly than when non-cluster roots were supplied. After treatment with labelled Gly (13C : 15N ratio = 1), lupin cluster roots had a 13C : 15N ratio of about 0·85 compared with 0·59 in labelled non-cluster roots. Rates of uptake of label from Gly did not differ between cluster and non-cluster roots of either species. The ratio of C : N and 13C : 15N in the plant increased in the order: labelled roots < rest of the root < shoot in both species, owing to an increasing proportion of 13C translocation. • Conclusions Cluster roots of lupin specifically acquired more intact Gly than non-cluster roots, whereas Gly uptake by the cluster and non-cluster roots of leucadendron was comparable. The uptake capacities of cluster roots are discussed in relation to spatial and morphological characteristics in the natural environment. PMID:16223736

Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

PubMed Central

2011-01-01

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods. PMID:22182234

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1986-05-01

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

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

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Pan; School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026; Xi, Zhaoyong

Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at threemore » different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1989-12-15

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 ofmore » 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.« less

Regional differences in lectin binding patterns of vestibular hair cells

NASA Technical Reports Server (NTRS)

Baird, Richard A.; Schuff, N. R.; Bancroft, J.

1994-01-01

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not stain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type 1 hair cells while labeling, as in the bullfrog, Type 2 hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

PubMed Central

Akasaka, K.; Li, H.; Yamada, H.; Li, R.; Thoresen, T.; Woodward, C. K.

1999-01-01

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place. PMID:10548039

Short communication: Comparison of 3 solid digesta passage markers in dairy cows.

PubMed

Lee, C; Hristov, A N

2014-03-01

This study investigated the usefulness of acid-detergent fiber-bound (15)N [acid detergent insoluble (ADI)-(15)N] as a solid digesta passage marker in dairy cows compared with chromium (Cr) and ytterbium (Yb) (as labeled fiber or forage, respectively). Intrinsically (ADI-(15)N) or extrinsically (Cr, Yb) labeled alfalfa hay was pulse-dosed intraruminally to 7 lactating dairy cows. Following marker administration, spot fecal samples were collected for up to 72 h for marker analyses. Urine and milk samples were also collected and analyzed for Yb and Cr. Fecal marker excretion data were processed using 2-compartment mathematical age-dependent/age-independent (Gn→G1) models. The rate of passage of the marker in the first, age-dependent compartment tended to be slower for Yb compared with Cr and ADI-(15)N, which resulted in a trend for longer mean retention time (MRT) in this compartment when Yb was used as a marker (19.0 h) compared with Cr and ADI-(15)N (14.5 and 13.9h, respectively). The rate constant of marker disappearance for the second or age-independent compartment tended to be greater for Yb compared with Cr and ADI-(15)N, which led to a shorter MRT of Yb in this compartment (15.6) versus ADI-(15)N (32.1) and Cr (24.8h). The cumulative MRT was greater for ADI-(15)N versus Cr and Yb (46.0, 39.3, and 34.4h, respectively). Total MRT of marker tended to be greater for ADI-(15)N than for Yb (46.6 vs. 36.6h, respectively). Urine and milk analyses data suggested no measurable losses of Yb along the digestive tract, but about 0.79% of Cr dosed intraruminally was secreted or excreted in milk and urine in the 48-h period following marker administration. Collectively, this study confirmed previous observations that ADI-(15)N can be used reliably as a solid digesta passage marker for ruminants, producing pre-duodenal and total-tract retention times similar to that of Cr-labeled fiber. Retention time in the age-independent compartment was underestimated when Yb was used as a marker, emphasizing the need to process forages to isolate fiber before labeling with Yb. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid 1

PubMed Central

Baldi, Bruce G.; Maher, Barbara R.; Slovin, Janet Pernise; Cohen, Jerry D.

1991-01-01

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[15N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[15N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants. PMID:16668112

Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-Methyladenine in Human Cells.

PubMed

Liu, Baodong; Liu, Xiaoling; Lai, Weiyi; Wang, Hailin

2017-06-06

DNA N 6 -methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope-labeled deoxynucleoside [ 15 N 5 ]-2'-deoxyadenosine ([ 15 N 5 ]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [ 15 N 5 ]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine 15 N, and further utilizes the purine salvage pathway to generate mainly both [ 15 N 4 ]-dA and [ 15 N 4 ]-2'-deoxyguanosine ([ 15 N 4 ]-dG) in mammalian genomes. However, [ 15 N 5 ]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [ 15 N 4 ]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.

Labelling and Self-Esteem: The Impact of Using Specific vs. Generic Labels

ERIC Educational Resources Information Center

Taylor, Laura Marie; Hume, Ian Robert; Welsh, Nikki

2010-01-01

The aim of this study is to investigate the relationship between being labelled either as having dyslexia or as having general special educational needs (SEN) and a child's self-esteem. Seventy-five children aged between 8 and 15 years categorised as having dyslexia (N = 26), as having general SEN (N = 26) or as having no learning difficulties (N…

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels.

PubMed

Virtanen, Otto L J; Purohit, Ashvini; Brugnoni, Monia; Wöll, Dominik; Richtering, Walter

2016-09-08

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Regional differences in lectin binding patterns of vestibular hair cells

NASA Technical Reports Server (NTRS)

Baird, R. A.; Schuff, N. R.; Bancroft, J.

1993-01-01

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylglucosamine (WGA), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not strain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type I hair cells while labeling, as in the bullfrog, Type II hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santos, J.A.

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

Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions.

PubMed

Hattori, Yoshikazu; Heidenreich, David; Ono, Yuki; Sugiki, Toshihiko; Yokoyama, Kei-Ichi; Suzuki, Ei-Ichiro; Fujiwara, Toshimichi; Kojima, Chojiro

2017-08-01

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15 N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19 F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19 F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19 F-labeling readily provided NMR detection of protein-drug and protein-protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19 F-labeling method was 3.5-fold more sensitive than 15 N-labeling, and could be combined with other chemical modification techniques such as lysine 13 C-methylation. 13 C-dimethylated- 19 F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

[The content-uniformity of dispensary-prepared rectal suppositories].

PubMed

Lüdde, H; Nestler, D

1990-01-01

Rectal suppositories, which are dispensed according prescription in small numbers up to N = 30, do not satisfy the demands in respect of content uniformity, if we consider the last N/10 poured ones. This by sedimentation caused problem is to be solved in increasing the amount of substances by N/10, so that you will get a safety-amount of 15% totally.

Simple and Robust N-Glycan Analysis Based on Improved 2-Aminobenzoic Acid Labeling for Recombinant Therapeutic Glycoproteins.

PubMed

Jeong, Yeong Ran; Kim, Sun Young; Park, Young Sam; Lee, Gyun Min

2018-03-21

N-glycans of therapeutic glycoproteins are critical quality attributes that should be monitored throughout all stages of biopharmaceutical development. To reduce both the time for sample preparation and the variations in analytical results, we have developed an N-glycan analysis method that includes improved 2-aminobenzoic acid (2-AA) labeling to easily remove deglycosylated proteins. Using this analytical method, 15 major 2-AA-labeled N-glycans of Enbrel ® were separated into single peaks in hydrophilic interaction chromatography mode and therefore could be quantitated. 2-AA-labeled N-glycans were also highly compatible with in-line quadrupole time-of-flight mass spectrometry (MS) for structural identification. The structures of 15 major and 18 minor N-glycans were identified from their mass values determined by quadrupole time-of-flight MS. Furthermore, the structures of 14 major N-glycans were confirmed by interpreting the MS/MS data of each N-glycan. This analytical method was also successfully applied to neutral N-glycans of Humira ® and highly sialylated N-glycans of NESP ® . Furthermore, the analysis data of Enbrel ® that were accumulated for 2.5 years demonstrated the high-level consistency of this analytical method. Taken together, the results show that a wide repertoire of N-glycans of therapeutic glycoproteins can be analyzed with high efficiency and consistency using the improved 2-AA labeling-based N-glycan analysis method. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashburn, D.A.; Garcia, K.; Hanners, J.L.

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{submore » 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.« less

Spin-echo based diagonal peak suppression in solid-state MAS NMR homonuclear chemical shift correlation spectra

NASA Astrophysics Data System (ADS)

Wang, Kaiyu; Zhang, Zhiyong; Ding, Xiaoyan; Tian, Fang; Huang, Yuqing; Chen, Zhong; Fu, Riqiang

2018-02-01

The feasibility of using the spin-echo based diagonal peak suppression method in solid-state MAS NMR homonuclear chemical shift correlation experiments is demonstrated. A complete phase cycling is designed in such a way that in the indirect dimension only the spin diffused signals are evolved, while all signals not involved in polarization transfer are refocused for cancellation. A data processing procedure is further introduced to reconstruct this acquired spectrum into a conventional two-dimensional homonuclear chemical shift correlation spectrum. A uniformly 13C, 15N labeled Fmoc-valine sample and the transmembrane domain of a human protein, LR11 (sorLA), in native Escherichia coli membranes have been used to illustrate the capability of the proposed method in comparison with standard 13C-13C chemical shift correlation experiments.

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

Treesearch

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

2014-01-01

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

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

Treesearch

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

2000-01-01

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

Pangamic Acid (Vitamin B15, Pangametin, Sopangamine)

PubMed Central

French, W. N.; Levi, Leo

1966-01-01

Pangamic acid is shown to be a mixture of variable composition. Criteria of identity and methods of analysis are described for five pharmaceutical dosage forms. Experimental results indicate that the products are not uniform in composition and that composition does not conform to label claims. No satisfactory preclinical drug application for any such preparation has so far been submitted to the Food and Drug Directorate. PMID:5295884

Studies of an Isolated 15N- 15N Spin Pair. Off-Angle Fast-Sample-Spinning NMR and Self-Consistent-Field Calculations for Diazo Systems

NASA Astrophysics Data System (ADS)

Challoner, Robin; Harris, Robin K.; Tossell, John A.

1997-05-01

An off-magic-angle spinning study of the nonassociated molecular solid, doubly15N-labeled 5-methyl-2-diazobenzenesulphonic acid hydrochloride (I) is reported. The validity of the off-magic-angle spinning approach under fast-spinning conditions is verified by average Hamiltonian theory. Ab initio SCF calculations were performed on the simpler molecule, C6H5N2+, to provide the shielding parameters, the dipolar coupling between the two nitrogen nuclei, and the electric field gradient existing at both the α-nitrogen and β-nitrogen sites. The calculated values are in good agreement with the shielding and effective dipolar coupling data elucidated in the present investigation, and with a previous study of the two singly15N-labeled isotopomers in which information concerning the electric field gradient at the α and β sites was deduced.

Synthesis and applications of [1-(15)N]-labeled 4,6-dimethyl-4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-oxide as a useful tool for mechanistic investigations.

PubMed

Sako, M; Yaekura, I; Oda, S; Hirota, K

2000-10-06

[1-(15)N]-Labeled 4,6-dimethyl-4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-oxide (1-(15)N1) was easily prepared by nitration of commercially available 6-amino-1,3-dimethyl-1H-pyrimidine-2,4-dione using 15N-enriched nitric acid followed by an intramolecular oxidative cyclization with iodosylbenzene diacetate under mild conditions. On the basis of the experimental results using 1-(15)N1, the formation of 8-phenyltheophylline (3), the 1,3-dimethylalloxazines (4: n = 0, 1), and 1,3,7,9-tetramethyl-1H,9H-pyrimido[5,4-g]pteridine-2,4,6,8-tetraone++ + (5) in the thermal reaction of the N-oxide 1 with benzylamine, aniline, or piperidine, and the generation of NO or NO-related species in the reaction with N-acetylcysteamine were reasonably explained by considering the initial attack of the employed nucleophiles on the 3a-position of 1.

Metabolism of α-linolenic acid during incubations with strained bovine rumen contents: products and mechanisms.

PubMed

Honkanen, Anne M; Leskinen, Heidi; Toivonen, Vesa; McKain, Nest; Wallace, R John; Shingfield, Kevin J

2016-06-01

Description of α-linolenic acid (cis-9,cis-12,cis-15-18 : 3, ALA) metabolism in the rumen is incomplete. Ruminal digesta samples were incubated with ALA and buffer containing water or deuterium oxide to investigate the products and mechanisms of ALA biohydrogenation. Geometric Δ9,11,15-18 : 3 isomers were the main intermediates formed from ALA. An increase in the n+1 isotopomers of Δ9,11,15-18 : 3 was due to 2H labelling at C-13. Isomers of Δ9,11,13-18 : 3, cis-7,cis-12,cis-15-18 : 3 and cis-8,cis-12,cis-15-18 : 3 were also formed. No increase in n+1 isotopomers of Δ7,12,15-18 : 3 or Δ8,12,15-18 : 3 was detected. Enrichment in n+2 isotopomers of 18 : 2 products indicated that ALA metabolism continued via the reduction of 18 : 3 intermediates. Isomers of Δ9,11,15-18 : 3 were reduced to Δ11,15-18 : 2 labelled at C-9 and C-13. ALA resulted in the formation of Δ11,13-18 : 2 and Δ12,14-18 : 2 containing multiple 2H labels. Enrichment of the n+3 isotopomer of Δ12,15-18 : 2 was also detected. Metabolism of ALA during incubations with rumen contents occurs by one of three distinct pathways. Formation of Δ9,11,15-18 : 3 appears to be initiated by H abstraction on C-13. Octadecatrienoic intermediates containing cis-12 and cis-15 double bonds are formed without an apparent H exchange with water. Labelling of Δ9,11,13-18 : 3 was inconclusive, suggesting formation by an alternative mechanism. These findings explain the appearance of several bioactive fatty acids in muscle and milk that influence the nutritional value of ruminant-derived foods.

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling1[W][OPEN

PubMed Central

Nelson, Clark J.; Alexova, Ralitza; Jacoby, Richard P.; Millar, A. Harvey

2014-01-01

Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis. PMID:25082890

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

PubMed Central

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

2015-01-01

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

Chinese Milk Vetch Improves Plant Growth, Development and 15N Recovery in the Rice-Based Rotation System of South China.

PubMed

Xie, Zhijian; He, Yaqin; Tu, Shuxin; Xu, Changxu; Liu, Guangrong; Wang, Huimin; Cao, Weidong; Liu, Hui

2017-06-15

Chinese milk vetch (CMV) is vital for agriculture and environment in China. A pot experiment combined with 15 N labeling (including three treatments: control, no fertilizer N and CMV; 15 N-labeled urea alone, 15 NU; substituting partial 15 NU with CMV, 15 NU-M) was conducted to evaluate the impact of CMV on plant growth, development and 15 NU recovery in rice-based rotation system. The 15 NU-M mitigated oxidative damage by increasing antioxidant enzymes activities and chlorophyll content while decreased malondialdehyde content in rice root and shoot, increased the biomass, total N and 15 N uptake of plant shoots by 8%, 12% and 39% respectively, thus inducing a noticeable increase of annual 15 N recovery by 77% versus 15 NU alone. Remarkable increases in soil NH 4 + and populations of bacteria, actinomycetes and azotobacter were obtained in legume-rice rotation system while an adverse result was observed in soil NO 3 - content versus fallow-rice. CMV as green manure significantly increased the fungal population which was decreased with cultivating CMV as cover crop. Therefore, including legume cover crop in rice-based rotation system improves plant growth and development, annual N conservation and recovery probably by altering soil nitrogen forms plus ameliorating soil microbial communities and antioxidant system which alleviates oxidative damages in plants.

Resource Utilization by Native and Invasive Earthworms and Their Effects on Soil Carbon and Nitrogen Dynamics in Puerto Rican Soils

Treesearch

Ching-Yu Huang; Grizelle Gonzalez; Paul F. Hendrix

2016-01-01

Resource utilization by earthworms affects soil C and N dynamics and further colonization of invasive earthworms. By applying 13C-labeled Tabebuia heterophylla leaves and 15N-labeled Andropogon glomeratus grass, we investigated resource utilization by three earthworm species (...

DIFFERENTIATION IN N15 UPTAKE AND THE ORGANIZATION OF AN ARCTIC TUNDRA PLANT COMMUNITY

EPA Science Inventory

We used N15 soil-labeling techniques to examine how the dominant species in a N-limited, tussock tundra plant community partitioned soil N, and how such partitioning may contribute to community organization. The five most abundant species were well differentiated with respect to...

Nitrogen Inputs via Nitrogen Fixation in Northern Plants and Soils

NASA Astrophysics Data System (ADS)

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

2015-12-01

Dominated by cold and often acidic water logged environments, mineralization of organic matter is slow in the majority of northern ecosystems. Measures of extractable ammonium and nitrate are generally low and can be undetectable in peat pore waters. Despite this apparent nitrogen limitation, many of these environments produce deep deposits of soil organic matter. Biological nitrogen fixation carried out by autotrophic and heterotrophic diazotrophs associated with cryptograms provides the majority of known nitrogen inputs in these northern ecosystems. Nitrogen fixation was assessed in a variety of northern soils within rhizospheres of dominant plant communities. We investigated the availability of this newly fixed nitrogen to the vascular plant community in nitrogen limited northern plant communities. We tracked nitrogen flow from 15N2 gas fixed in Sphagnum mosses into tissues of two native vascular plant species, boreal cranberry (Vaccinium oxycoccus) and black spruce (Picea mariana). 15N-labeled Sphagnum microcosms were grown within variable mesh size exclusion/inclusion fabrics in a nitrogen addition experiment in situ in order to investigate the role of mycorrhizal fungi in the uptake of newly fixed nitrogen. Up to 24% of daily fixed 15N label was transferred to vascular plant tissues during 2 months. Nitrogen addition resulted in decreased N2 fixation rates; however, with higher nitrogen availability there was a higher rate of 15N label uptake into the vascular plants, likely the result of increased production of dissolved organic nitrogen. Reliance on mycorrhizal networks for nitrogen acquisition was indicated by nitrogen isotope fractionation patterns. Moreover, N2 fixation activities in mosses were stimulated when vascular plants were grown in moss microcosms versus "moss only" treatments. Results indicate that bog vascular plants may derive considerable nitrogen from atmospheric N2 biologically fixed within Sphagnum mosses. This work demonstrates that diazotroph-mediated 15N labeling is a viable technique for tracking nitrogen flow without altering form and concentration of native nitrogen pools in a nitrogen limited ecosystem.

Combining Metabolic ¹⁵N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

PubMed

Thomas, Martin; Huck, Nicola; Hoehenwarter, Wolfgang; Conrath, Uwe; Beckers, Gerold J M

2015-01-01

In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. This is particularly true for plants in which the presence of secondary metabolites and endogenous compounds, the overabundance of ribulose-1,5-bisphosphate carboxylase and other components of the photosynthetic apparatus, and the concurrent difficulties in protein extraction necessitate two-step phosphoprotein/phosphopeptide enrichment strategies (Nakagami et al., Plant Cell Physiol 53:118-124, 2012).Approaches for label-free peptide quantification are advantageous due to their low cost and experimental simplicity, but they lack precision. These drawbacks can be overcome by metabolic labeling of whole plants with heavy nitrogen ((15)N) which allows combining two samples very early in the phosphoprotein enrichment workflow. This avoids sample-to-sample variation introduced by the analytical procedures and it results in robust relative quantification values that need no further standardization. The integration of (15)N metabolic labeling into tandem metal-oxide affinity chromatography (MOAC) (Hoehenwarter et al., Mol Cell Proteomics 12:369-380, 2013) presents an improved and highly selective approach for the identification and accurate site-specific quantification of low-abundance phosphoproteins that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope ((15)N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while (15)N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.

Magnetizable stent-grafts enable endothelial cell capture

NASA Astrophysics Data System (ADS)

Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

Magnetizable stent-grafts enable endothelial cell capture.

PubMed

Tefft, Brandon J; Uthamaraj, Susheil; Harburn, J Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

On the problem of resonance assignments in solid state NMR of uniformly 15N, 13C-labeled proteins

NASA Astrophysics Data System (ADS)

Tycko, Robert

2015-04-01

Determination of accurate resonance assignments from multidimensional chemical shift correlation spectra is one of the major problems in biomolecular solid state NMR, particularly for relative large proteins with less-than-ideal NMR linewidths. This article investigates the difficulty of resonance assignment, using a computational Monte Carlo/simulated annealing (MCSA) algorithm to search for assignments from artificial three-dimensional spectra that are constructed from the reported isotropic 15N and 13C chemical shifts of two proteins whose structures have been determined by solution NMR methods. The results demonstrate how assignment simulations can provide new insights into factors that affect the assignment process, which can then help guide the design of experimental strategies. Specifically, simulations are performed for the catalytic domain of SrtC (147 residues, primarily β-sheet secondary structure) and the N-terminal domain of MLKL (166 residues, primarily α-helical secondary structure). Assuming unambiguous residue-type assignments and four ideal three-dimensional data sets (NCACX, NCOCX, CONCA, and CANCA), uncertainties in chemical shifts must be less than 0.4 ppm for assignments for SrtC to be unique, and less than 0.2 ppm for MLKL. Eliminating CANCA data has no significant effect, but additionally eliminating CONCA data leads to more stringent requirements for chemical shift precision. Introducing moderate ambiguities in residue-type assignments does not have a significant effect.

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.

PubMed

Lechene, Claude; Hillion, Francois; McMahon, Greg; Benson, Douglas; Kleinfeld, Alan M; Kampf, J Patrick; Distel, Daniel; Luyten, Yvette; Bonventre, Joseph; Hentschel, Dirk; Park, Kwon Moo; Ito, Susumu; Schwartz, Martin; Benichou, Gilles; Slodzian, Georges

2006-01-01

Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

PubMed

Kim, Kyoung-Jin; Kim, Yoon-Woo; Kim, Yun-Gon; Park, Hae-Min; Jin, Jang Mi; Hwan Kim, Young; Yang, Yung-Hun; Kyu Lee, Jun; Chung, Junho; Lee, Sun-Gu; Saghatelian, Alan

2015-01-01

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers.

Defining the interaction of human soluble lectin ZG16p and mycobacterial phosphatidylinositol mannosides

PubMed Central

Ikeda, Akemi; Kojima-Aikawa, Kyoko; Taniguchi, Naoyuki; Varón Silva, Daniel; Feizi, Ten; Seeberger, Peter H.; Yamaguchi, Yoshiki

2018-01-01

ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analyses of the interactions of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs), using glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation Transfer Difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interacts with ZG16p using the mannose residues. Binding site of PIMs is identified by chemical shift perturbation experiments using uniformly 15N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan, which would help to consider the physiological role of ZG16p. PMID:25919894

Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons.

PubMed

Bak, Lasse K; Johansen, Maja L; Schousboe, Arne; Waagepetersen, Helle S

2012-09-01

Synthesis of neuronal glutamate from α-ketoglutarate for neurotransmission necessitates an amino group nitrogen donor; however, it is not clear which amino acid(s) serves this role. Thus, the ability of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, to act as amino group nitrogen donors for synthesis of vesicular neurotransmitter glutamate was investigated in cultured mouse cerebellar (primarily glutamatergic) neurons. The cultures were superfused in the presence of (15) N-labeled BCAAs, and synaptic activity was induced by pulses of N-methyl-D-aspartate (300 μM), which results in release of vesicular glutamate. At the end of the superfusion experiment, the vesicular pool of glutamate was released by treatment with α-latrotoxin (3 nM, 5 min). This experimental paradigm allows a separate analysis of the cytoplasmic and vesicular pools of glutamate. Amount and extent of (15) N labeling of intracellular amino acids plus vesicular glutamate were analyzed employing HPLC and LC-MS analysis. Only when [(15) N]valine served as precursor did the labeling of both cytoplasmic and vesicular glutamate increase after synaptic activity. In addition, only [(15) N]valine was able to maintain the amount of vesicular glutamate during synaptic activity. This indicates that, among the BCAAs, only valine supports the increased need for synthesis of vesicular glutamate. Copyright © 2012 Wiley Periodicals, Inc.

75 FR 71344 - Uniform Compliance Date for Food Labeling Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-23

... DEPARTMENT OF AGRICULTURE Food Safety and Inspection Service 9 CFR Parts 317 and 381 [Docket No. FSIS-2010-0031] RIN 0583-AD Uniform Compliance Date for Food Labeling Regulations AGENCY: Food Safety and Inspection Service, USDA. ACTION: Final rule. SUMMARY: The Food Safety and Inspection Service...

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism.

PubMed

Moriya, Shunsuke; Iwasaki, Kaori; Samejima, Keijiro; Takao, Koichi; Kohda, Kohfuku; Hiramatsu, Kyoko; Kawakita, Masao

2012-10-20

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N(1)-acetylspermidine (N(1)AcSpd), N(8)-acetylspermidine (N(8)AcSpd), N(1)-acetylspermine, N(1),N(8)-diacetylspermidine, and N(1),N(12)-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N(1)AcSpd and N(8)AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with (13)C(2)-N(1)AcSpd and (13)C(2)-N(8)AcSpd which have the (13)C(2)-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N(1)-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N(1)-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-(15)N(3)]-N(1)-acetylspermine and [1,4,8-(15)N(3)]spermidine ((15)N(3)-Spd), respectively; for SMO, [1,4,8,12-(15)N(4)]spermine and (15)N(3)-Spd, respectively; and for SSAT, (15)N(3)-Spd and [1,4,8-(15)N(3)]-N(1)-acetylspermidine, respectively. Copyright © 2012 Elsevier B.V. All rights reserved.

Application of capillary gas chromatography-reaction interface/mass spectrometry to the selective detection of sup 13 C-, sup 15 N-, sup 2 H-, and sup 14 C-labeled drugs and their metabolites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chace, D.H.

1989-01-01

A novel reaction interface/mass spectrometer (RIMS) technique has been applied to the selective detection of {sup 13}C-, {sup 15}N-, {sup 2}H-, and {sup 14}C-labeled phenytoin and its metabolites in urine following separation by capillary gas chromatography. The microwave-powered reaction interface converts materials from their original forms into small molecules whose mass spectra serve to identify and quantify the nuclides. The presence of each element is followed by monitoring the isotopic variants of CO{sub 2}, NO, H{sub 2}, or CH{sub 4} that are produced by the reaction interface. Chromatograms showing only enriched {sup 13}C and {sup 15}N were produced using themore » net {sup 13}CO{sub 2} or {sup 15}NO signal derived by subtracting the abundance of naturally occurring isotopes from the observed M + 1 signal. When hydrogen was used as a reactant gas, a selective chromatogram of {sup 2}H (D) was obtained by measuring HD at m/Z 3.0219, and a chromatogram showing {sup 14}C was obtained by measuring {sup 14}CH{sub 4} at m/Z 18.034 with a high resolution. For a stable isotope detection, metabolites representing less than 1.5% of the total labeled compounds could be detected in the chromatogram. Detection limits of 170 pCi/mL (34 pCi on column that is equivalent to 187 pg) of a {sup 14}C- labeled metabolite was detected. To identify many of these labeled peaks (metabolites), the chromatographic analysis was repeated with the reaction interface turned off and mass spectra obtained at the retention times found in the RIMS experiment. In addition to the ability of GC-RIMS to detect the presence of {sup 13}C-, {sup 15}N-, and {sup 2}H- (D), it can also quantify the level of enrichment. Enrichment of {sup 13}C and {sup 15}N is quantified by measuring the ratio of excess {sup 13}CO{sub 2} to total {sup 12}CO{sub 2} or excess {sup 15}NO to total {sup 14}NO.« less

In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

PubMed

Alonso-Pernas, Pol; Bartram, Stefan; Arias-Cordero, Erika M; Novoselov, Alexey L; Halty-deLeon, Lorena; Shao, Yongqi; Boland, Wilhelm

2017-01-01

The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer ( Melolontha hippocastani ), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13 C cellulose and 15 N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13 C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15 N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13 C cellulose- and 15 N urea labeled bacteria. The incorporation of 15 N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani , this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

Preferences for different nitrogen forms by coexisting plant species and soil microbes.

PubMed

Harrison, Kathryn A; Bol, Roland; Bardgett, Richard D

2007-04-01

The growing awareness that plants might use a variety of nitrogen (N) forms, both organic and inorganic, has raised questions about the role of resource partitioning in plant communities. It has been proposed that coexisting plant species might be able to partition a limited N pool, thereby avoiding competition for resources, through the uptake of different chemical forms of N. In this study, we used in situ stable isotope labeling techniques to assess whether coexisting plant species of a temperate grassland (England, UK) display preferences for different chemical forms of N, including inorganic N and a range of amino acids of varying complexity. We also tested whether plants and soil microbes differ in their preference for different N forms, thereby relaxing competition for this limiting resource. We examined preferential uptake of a range of 13C15N-labeled amino acids (glycine, serine, and phenylalanine) and 15N-labeled inorganic N by coexisting grass species and soil microbes in the field. Our data show that while coexisting plant species simultaneously take up a variety of N forms, including inorganic N and amino acids, they all showed a preference for inorganic N over organic N and for simple over the more complex amino acids. Soil microbes outcompeted plants for added N after 50 hours, but in the long-term (33 days) the proportion of added 15N contained in the plant pool increased for all N forms except for phenylalanine, while the proportion in the microbial biomass declined relative to the first harvest. These findings suggest that in the longer-term plants become more effective competitors for added 15N. This might be due to microbial turnover releasing 15N back into the plant-soil system or to the mineralization and subsequent plant uptake of 15N transferred initially to the organic matter pool. We found no evidence that soil microbes preferentially utilize any of the N forms added, despite previous studies showing that microbial preferences for N forms vary over time. Our data suggest that coexisting plants can outcompete microbes for a variety of N forms, but that such plant species show similar preferences for inorganic over organic N.

Amyloid Hydrogen Bonding Polymorphism Evaluated by (15)N{(17)O}REAPDOR Solid-State NMR and Ultra-High Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

PubMed

Wei, Juan; Antzutkin, Oleg N; Filippov, Andrei V; Iuga, Dinu; Lam, Pui Yiu; Barrow, Mark P; Dupree, Ray; Brown, Steven P; O'Connor, Peter B

2016-04-12

A combined approach, using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and solid-state NMR (Nuclear Magnetic Resonance), shows a high degree of polymorphism exhibited by Aβ species in forming hydrogen-bonded networks. Two Alzheimer's Aβ peptides, Ac-Aβ(16-22)-NH2 and Aβ(11-25), selectively labeled with (17)O and (15)N at specific amino acid residues were investigated. The total amount of peptides labeled with (17)O as measured by FTICR-MS enabled the interpretation of dephasing observed in (15)N{(17)O}REAPDOR solid-state NMR experiments. Specifically, about one-third of the Aβ peptides were found to be involved in the formation of a specific >C═(17)O···H-(15)N hydrogen bond with their neighbor peptide molecules, and we hypothesize that the rest of the molecules undergo ± n off-registry shifts in their hydrogen bonding networks.

Positron emitting nuclides and their synthetic incorporation in radiopharmaceuticals. [Labeled with /sup 11/C, /sup 13/N, and /sup 18/F

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, J.S.

/sup 11/C, /sup 13/N, and /sup 15/O has potential applicability to the study of metabolism in humans. Problems in the synthesis of radiopharmaceuticals labeled with /sup 11/C, /sup 13/N, and /sup 18/F are described: quality control, radiation exposure, carboxylic acids, glucose, amines, amino acids, nitrosources, fluoroethanol. 54 references. (DLC)

Overlapping-image multimode interference couplers with a reduced number of self-images for uniform and nonuniform power splitting

NASA Astrophysics Data System (ADS)

Bachmann, M.; Besse, P. A.; Melchior, H.

1995-10-01

Overlapping-image multimode interference (MMI) couplers, a new class of devices, permit uniform and nonuniform power splitting. A theoretical description directly relates coupler geometry to image intensities, positions, and phases. Among many possibilities of nonuniform power splitting, examples of 1 \\times 2 couplers with ratios of 15:85 and 28:72 are given. An analysis of uniform power splitters includes the well-known 2 \\times N and 1 \\times N MMI couplers. Applications of MMI couplers include mode filters, mode splitters-combiners, and mode converters.

Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.

PubMed

Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica M; Peters, Thomas

2017-07-04

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H, 15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H, 15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[ 13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2017-01-01

Ecological treatments are effective for treating agricultural wastewater. In this study, wetland microcosms vegetated with Myriophyllum aquaticum were designed for nitrogen (N) removal from two strengths of swine wastewater, and 15 N-labelled ammonium (NH 4 + -N) was added to evaluate the dominant NH 4 + -N removal pathway. The results showed that 98.8% of NH 4 + -N and 88.3% of TN (TN: 248.6 mg L -1 ) were removed from low-strength swine wastewater (SW1) after an incubation of 21 days, with corresponding values for high-strength swine wastewater (SW2) being 99.2% of NH 4 + -N and 87.8% of TN (TN: 494.9 mg L -1 ). Plant uptake and soil adsorption respectively accounted for 24.0% and 15.6% of the added 15 N. Meanwhile, above-ground tissues of M. aquaticum had significantly higher biomass and TN content than below-ground (P < 0.05). 15 N mass balance analysis indicated that gas losses contributed 52.0% to the added 15 N, but the N 2 O flux constituted only 7.5% of total gas losses. The dynamics of NO 3 - -N and N 2 O flux revealed that strong nitrification and denitrification occurred in M. aquaticum microcosms, which was a dominant N removal pathway. These findings demonstrated that M. aquaticum could feasibly be used to construct wetlands for high N-loaded animal wastewater treatment. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Absorption and translocation of nitrogen in rhizomes of Leymus chinensis.

PubMed

Liu, Hongsheng; Liu, Huajie; Song, Youhong

2011-03-15

Leymus chinensis is a dominant species in the Inner Mongolia steppe, northern China. Plant growth in northern China grassland is often limited by low soil nitrogen availability. The objective of this study is to investigate whether rhizomes of Leymus chinensis are involved in the contribution of N uptake. The N concentration, (15)N concentration and (15)N proportion in roots, rhizomes and shoots after 48 h exposure of roots (L(root)) and rhizomes (L(rhizo)) separately and roots and rhizomes together (L(r+r)) to 0.1 mM (15)NH (4)(15)NO(3) solution were measured using root-splitting equipment and stable isotope ((15)N) techniques, respectively. The N content and dry mass were not affected by the labeling treatment. In contrast, the (15)N concentration in shoots, rhizomes and roots was significantly increased by the labeling in rhizomes, indicating that the inorganic nitrogen was absorbed via rhizomes from the solution and can be transported to other tissues, with preference to shoots rather than roots. Meanwhile, the absolute N absorption and translocation among compartments were also calculated. The N absorption via rhizomes was much smaller than via roots; however, the uptake efficiency per surface unit via rhizomes was greater than via roots. The capacity and high efficiency to absorb N nutrient via rhizomes enable plants to use transient nutrient supplies in the top soil surface. Copyright © 2011 John Wiley & Sons, Ltd.

Modification and integration of JSW cyclotron GAS targets at the national institutes of health cyclotron facility

NASA Astrophysics Data System (ADS)

Finn, R.; Plascjak, P.; Sheh, Y.; Yamashita, Y.; Yoshida, H.; Adams, R.; Simpson, N.; Larson, S.

1987-04-01

The Cyclotron staff at the National Institutes of Health is involved in a comprehensive radionuclide preparation program which culminates with the formulation of numerous requested short-lived radiopharmaceutical agents for clinical evaluation. The existence of two cyclotrons and the requests for cyclotron-produced radionuclides, principally short-lived positron-emitting ones, necessitates an efficient and cost-effective program. The clinical need for 15O labelled water exemplifies the modification and effective coupling of two supplied gas target systems without detriment to either individual product. 15O labeled oxygen, produced from the 14N(d,n) 15O nuclear reaction, is combined with the target gas for 11C labelled cyanide production through standard fittings to achieve the chemical oxidation. The system allows an "on-line" product of extremely high yield and excellent radionuclidic purity. The operational characteristics of the redesigned commercial cyclotron targetry system and the radiochemical considerations are presented.

Surface proteins and the formation of biofilms by Staphylococcus aureus.

PubMed

Kim, Sung Joon; Chang, James; Rimal, Binayak; Yang, Hao; Schaefer, Jacob

2018-03-01

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [ 15 N]glycine and l-[1- 13 C]threonine, or in separate experiments, by (ii) l-[2- 13 C, 15 N]leucine. We then measured 13 C- 15 N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15 N isotopic enrichments and from the routing of 13 C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion. Copyright © 2017 Elsevier B.V. All rights reserved.

Sediment DIN fluxes and preferential recycling of benthic microalgal nitrogen in a shallow macrotidal estuary

USGS Publications Warehouse

Tobias, C.; Giblin, A.; McClelland, J.; Tucker, J.; Peterson, B.

2003-01-01

Sediment-water fluxes of NH4+, NO3-, dissolved inorganic carbon, and O2 were measured in cores collected from the upper Rowley River estuary, Massachusetts, and used to calculate rates of organic nitrogen (N) mineralization, nitrification, and coupled and direct denitrification (DNF). The cores contained 15N label in benthic microalgae (BMA) and in NO3- in the overlying water as a result of an ongoing whole-estuary 15NO 3- enrichment study (NISOTREX II). The tracer allowed for estimation of gross NO3- regeneration in sediments and the contribution of BMA derived N to total mineralization. The mean mineralization rate between sites was 16.0 ?? 2.0 mmol N m-2 d-1. Approximately 13 to 56% of the mineralized N was nitrified at rates ranging from 1.8 to 10.1 mmol N m-2 d-1. Total denitrification was dominated by direct DNF (3.6 mmol N m-2 d -1) furthest upstream, where NO3- concentrations were highest. Coupled DNF was most important (8.0 mmol N m -2 d-1) in the sediments with high nitrification and low water column NO3-. A gross NO3- flux from sediments to water of 0.9 to 2.1 mmol N m-2 d-1 was estimated from the isotope dilution of ??15NO 3- in the overlying water of the cores. The isotope dilution seen in the cores was also detected as a deviation from conservative ??15NO3- mixing along estuarine transects. Incorporation of this NO3- regeneration into the DNF calculations effectively increased the estimate of direct DNF by up to 50% and decreased the coupled DNF estimate by up to 220%. Increasing ?? 15NH4+ in the water of the cores indicated that the 15N-labelled BMA were preferentially mineralized over bulk sediment organic N. Additional 15N enrichments in the sediment bacterial biomarker diaminopimelic acid showed a link among 15N-labeled BMA, active bacteria, and 15NH 4+released to the overlying water. Based on ?? 15NH4+ enrichments in the cores, BMA accounted for approximately 50 to 100% of the N mineralized. An isotopic enrichment of ??15NH4+ above background in the estuary was observed at a magnitude consistent with the core-based rates of BMA mineralization. These results provide further evidence that BMA are not unidirectional sinks for water column-dissolved organic nitrogen, but instead act to turn over N between sediments and estuarine water on the scale of days.

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation.

PubMed

Zhou, Zhengyuan; Vaidyanathan, Ganesan; McDougald, Darryl; Kang, Choong Mo; Balyasnikova, Irina; Devoogdt, Nick; Ta, Angeline N; McNaughton, Brian R; Zalutsky, Michael R

2017-12-01

Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[ 18 F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([ 18 F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [ 18 F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [ 18 F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[ 18 F]RL-I conjugates. [ 18 F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [ 125 I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [ 18 F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [ 125 I]SGMIB-2Rs15d. Uptake of [ 18 F]RL-I-2Rs15d in subcutaneous xenografts was 16-20 %ID/g over 1-3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [ 18 F]RL-I-2Rs15d but not with [ 18 F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [ 18 F]RL-I-2Rs15d and [ 18 F]RL-I-5F7. Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

75 FR 78155 - Uniform Compliance Date for Food Labeling Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-15

... labeling requirements to minimize the economic impact of label changes. On December 8, 2008, FDA... dates of these labeling changes were not coordinated, the cumulative economic impact on the food... for an orderly and economical industry adjustment to new labeling requirements by allowing sufficient...

13C-13C dipolar recoupling under very fast magic angle spinning in solid-state nuclear magnetic resonance: Applications to distance measurements, spectral assignments, and high-throughput secondary-structure determination

NASA Astrophysics Data System (ADS)

Ishii, Yoshitaka

2001-05-01

A technique is presented to recouple homonuclear dipolar couplings between dilute spin pairs such as 13C-13C systems under very fast magic angle spinning (MAS) in solid-state nuclear magnetic resonance (NMR) spectroscopy. The presented technique, finite pulse rf driven recoupling (fpRFDR), restores homonuclear dipolar interactions based on constructive usage of finite pulse-width effects in a phase- and symmetry-cycled π-pulse train in which a rotor-synchronous π pulse is applied every rotation period. The restored effective dipolar interaction has the form of a zero-quantum dipolar Hamiltonian for static solids, whose symmetry in spin space is different from that obtained by conventional rf driven recoupling (RFDR) techniques. It is demonstrated that the efficiency of recoupling by fpRFDR is not strongly dependent on chemical shift differences or resonance offsets in contrast to previous recoupling methods under very fast MAS. To realize distance measurements without effects of spin relaxation, a constant-time version of fpRFDR (CT-fpRFDR) is introduced, in which the effective evolution period is varied by refocusing dipolar evolution with a rotor-synchronized solid echo while the total recoupling period is kept constant. From CT-fpRFDR experiments at a spinning speed of 30.3 kHz in a field of 17.6 T, the 13C-13C distance of [1-13C]Ala-[1-13C]Gly-Gly was determined to be 3.27 Å, which agrees well with the value of 3.20 Å obtained by x-ray diffraction. Also, two-dimensional (2D) 13C/13C chemical-shift correlation NMR spectrum in a field of 9.4 T was obtained with fpRFDR for fibrils of the segmentally 13C- and 15N-labeled Alzheimer's β-Amyloid fragments, Aβ16-22 (residues 16-22 taken from the 40-residue Aβ peptide) in which Leu-17 through Ala-21 are uniformly 13C- and 15N-labeled. Most 13C resonances for the main chain as well as for the side chains are assigned based on 2D 13C/13C chemical-shift correlation patterns specific to amino-acid types. Examination of the obtained 13C chemical shifts revealed the formation of β-strand across the entire molecule of Aβ16-22. Possibility of high throughput determination of global main-chain structures based on 13C shifts obtained from 2D 13C/13C chemical-shift correlation under very fast MAS is also discussed for uniformly/segmentally 13C-labeled protein/peptide samples.

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

DTIC Science & Technology

1990-05-22

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

IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells

PubMed Central

Orlando, Ron; Lim, Jae-Min; Atwood, James A.; Angel, Peggi M.; Fang, Meng; Aoki, Kazuhiro; Alvarez-Manilla, Gerardo; Moremen, Kelley W.; York, William S.; Tiemeyer, Michael; Pierce, Michael; Dalton, Stephen; Wells, Lance

2012-01-01

Robust quantification is an essential component of comparative –omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, has not been described. Here we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-15N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we demonstrate that culturing mouse embryonic stems cells for 72 hours in the presence of amide-15N-Gln media results in nearly complete incorporation of 15N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is a simple to implement, yet powerful quantitative tool for the glycomics toolbox. PMID:19449840

Time-reversal-based SU(2) x Sn scalar invariants as (Lie Algebraic) group measures: a structured overview of generalised democratic-recoupled, uniform non-Abelian [AX]n NMR spin systems, as abstract [Formula: see text] chain networks.

PubMed

Temme, F P

2004-03-01

The physics of dual group scalar invariants (SIs) as (Lie algebraic) group measures (L-GMs) and its significance to non-Abelian NMR spin systems motivates this overview of uniform general-2n [AX](2n) spin evolution, which represents an extensive addendum to Corio's earlier (essentially restricted) view of Abelian spin system SU(2)-based SI-cardinalities. The [Formula: see text] values in [J. Magn. Reson., 134 (1998) 131] arise from strictly linear recoupled time-reversal invariance (TRI) models. In contrast, here we discuss the physical significance of an alternative polyhedral combinatorics approach to democratic recoupling (DR), a property inherent in both the TRI and statistical sampling. Recognition of spin ensemble SIs as being L-GMs over isomorphic algebras is invaluable in many DR-based NMR problems. Various [AX]n model spin systems, including the [AX]3 bis odd-odd parity spin system, are examined as direct applications of these L-GM- and combinatorial-based SI ideas. Hence in place of /SI/=15 (implied by Corio's [Formula: see text] approach), the bis 3-fold spin system cardinality is seen now as constrained to a single invariant on an isomorphic product algebra under L-GMs, in accord with the subspectral analysis of Jones et al. [Canad. J. Chem., 43 (1965) 683]. The group projective ideas cited here for DR (as cf. to graph theoretic views) apply to highly degenerate non-Abelian problems. Over dual tensorial bases, they define models of spin dynamical evolution whose (SR) quasiparticle superboson carrier (sub)spaces are characterised by SIs acting as explicit auxiliary labels [Physica, A198 (1993) 245; J. Math. Chem., 31 (2002) 281]. A deeper [Formula: see text] network-based view of spin-alone space developed in Balasubramanian's work [J. Chem. Phys., 78 (1983) 6358] is especially important, (e.g.) in the study of spin waves [J. Math. Chem., 31 (2002) 363]. Beyond the specific NMR SIs derived here, there are DR applications where a sporadic, still higher, 2n-fold regular uniform spin ensemble exhibits a topological FG duality to some known modest /SI/(2i<2n) cardinality--in principle providing for the (sparce) existence of other /SI/(2n) DR-based values.

Characterization of biodegradation intermediates of nonionic surfactants by MALDI-MS. 2. Oxidative biodegradation profiles of uniform octylphenol polyethoxylate in 18O-labeled water.

PubMed

Sato, Hiroaki; Shibata, Atsushi; Wang, Yang; Yoshikawa, Hiromichi; Tamura, Hiroto

2003-01-01

This paper reports the characterization of the biodegradation intermediates of octylphenol octaethoxylate (OP(8)EO) by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The biodegradation test study was carried out in a pure culture (Pseudomonas putida S-5) under aerobic conditions using OP(8)EO as the sole carbon source and (18)O-labeled water as an incubation medium. In the MALDI-MS spectra of biodegraded samples, a series of OP(n)EO molecules with n = 2-8 EO units and their corresponding carboxylic acid products (OP(n)EC) were observed. The use of purified OP(8)EO enabled one to distinguish the shortened OPEO molecules as biodegradation intermediates. Furthermore, the formation of OP(8)EC (the oxidized product of OP(8)EO) supported the notion that terminal oxidation is a step in the biodegradation process. When biodegradation study was carried out in (18)O-labeled water, incorporation of (18)O atoms into the carboxyl group was observed for OPEC, while no incorporation was observed for the shortened OPEO products. These results could provide some rationale to the biodegradation mechanism of alkylphenol polyethoxylates.

The evaluation of new and isotopically labeled isoindoline nitroxides and an azaphenalene nitroxide for EPR oximetry

PubMed Central

Khan, Nadeem; Blinco, James P.; Bottle, Steven E.; Hosokawa, Kazuyuki; Swartz, Harold M.; Micallef, Aaron S.

2011-01-01

Isoindoline nitroxides are potentially useful probes for viable biological systems, exhibiting low cytotoxicity, moderate rates of biological reduction and favorable Electron Paramagnetic Resonance (EPR) characteristics. We have evaluated the anionic (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl; CTMIO), cationic (5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethylisoindolin-2-yloxyl iodide, QATMIO) and neutral (1,1,3,3-tetramethylisoindolin-2-yloxyl; TMIO) nitroxides and their isotopically labeled analogues (2H12- and/or 2H12-15N-labeled) as potential EPR oximetry probes. An active ester analogue of CTMIO, designed to localize intracellularly, and the azaphenalene nitroxide 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO) were also studied. While the EPR spectra of the unlabeled nitroxides exhibit high sensitivity to O2 concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O2 sensitivity. Labeling the nitroxides with 15N increased the signal intensity and this may be useful in decreasing the detection limits for in vivo measurements. The active ester nitroxide showed approximately 6% intracellular localization and low cytotoxicity. The EPR spectra of TMAO nitroxide indicated an increased rigidity in the nitroxide ring, due to dibenzo-annulation. PMID:21665499

Adsorption and wetting characterization of hydrophobic SBA-15 silicas.

PubMed

Bernardoni, Frank; Fadeev, Alexander Y

2011-04-15

This work describes adsorption and wetting characterization of hydrophobic ordered mesoporous silicas (OMSs) with the SBA-15 motif. Three synthetic approaches to prepare hydrophobic SBA-15 silicas were explored: grafting with (1) covalently-attached monolayers (CAMs) of C(n)H(2)(n+1)Si(CH(3))(2)N(CH(3))(2), (2) self-assembled monolayers (SAMs) of C(n)H(2)(n+1)Si(OEt)(3), and (3) direct ("one-pot") co-condensation of TEOS with C(n)H(2)(n+1)Si(OEt)(3) in presence of P123 (n=1-18). The materials prepared were characterized by nitrogen adsorption, TEM, and chemical analysis. The surface properties of the materials were assessed by water contact angles (CAs) and by BET C constants. The results showed that, while loadings of the alkyl groups (%C) were comparable, the surface properties and pore ordering of the materials prepared through different methods were quite different. The best quality hydrophobic surfaces were prepared for SBA-15 grafted with CAMs of alkylsilanes. For these materials, the water CAs were above ∼120°/100° (adv/rec) and BET C constants were in the range of ∼15-25, indicating uniform low-energy surfaces of closely packed alkyl groups on external and internal surfaces of the pores respectively. Moreover, surfaces grafted with the long-chained (C(12)-C(18)) silanes showed super-hydrophobic behavior (CAs∼150-180°) and extremely low adhesion for water. The pore uniformity of parental SBA-15 was largely preserved and the pore volume and pore diameter were consistent with the formation of a single layer of alkylsilyl groups inside the pores. Post-synthesis grafting of SBA-15 with SAMs worked not as well as CAMs: the surfaces prepared demonstrated lower water CAs and higher BET C constants, thereby indicating a small amount of accessible polar groups (Si-OH) related to packing constrains for SAMs supported on highly curved surfaces of mesopores. The co-condensation method produced substantially more disordered materials and less hydrophobic surfaces than any of the grafting methods. The surfaces of these materials showed low water CAs and high BET C constants (∼100-200) thereby demonstrating a non-uniform surface coverage and presence of unmodified silica. It is concluded that CAMs chemistry is the most efficient approach in preparation of the functionalized OMS materials with uniform surfaces and pores. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-06-01

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

Evidence for substantial forestry canopy processing of nitrogen deposition using isotopic tracer experiments in low deposition conditions

NASA Astrophysics Data System (ADS)

Ferraretto, Daniele; Heal, Kate

2017-04-01

Temperate forest ecosystems are significant sinks for nitrogen deposition (Ndep) yielding benefits such as protection of waterbodies from eutrophication and enhanced sequestration of atmospheric CO2. Previous studies have shown evidence of biological nitrification and Ndep processing and retention in forest canopies. However, this was reported only at sites with high environmental or experimentally enhanced rates of Ndep (˜18 kg N ha-1 y-1) and has not yet been demonstrated in low Ndep environments. We have used bulk field hydrochemical measurements and labelled isotopic experiments to assess canopy processing in a lower Ndep environment (˜7 kg N ha-1 year-1) at a Sitka spruce plantation in Perthshire, Scotland, representing the dominant tree species (24%) in woodlands in Great Britain. Analysis of 4.5 years of measured N fluxes in rainfall (RF) and fogwater onto the canopy and throughfall (TF) and stemflow (SF) below the canopy suggests strong transformation and uptake of Ndep in the forest canopy. Annual canopy Ndep uptake was ˜4.7 kg N ha-1 year-1, representing 60-76% of annual Ndep. To validate these plot-scale results and track N uptake within the forest canopy in different seasons, double 15N-labelled NH4NO3 (98%) solution was sprayed in summer and winter onto the canopy of three trees at the measurement site. RF, TF and SF samples have been collected and analysed for 15NH4 and 15NO3. Comparing the amount of labelled N recovered under the sample trees with the measured δ15N signal is expected to provide further evidence of the role of forest canopies in actively processing and retaining atmospheric N deposition.

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

NASA Astrophysics Data System (ADS)

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

2014-12-01

Metabolic rates of microbial communities constitute a key physiological parameter for understanding the in situ growth constraints for life in any environment. Isotope labeling techniques provide a powerful approach for measuring such biological activity, due to the use of isotopically enriched substrate tracers whose incorporation into biological materials can be detected with high sensitivity by isotope-ratio mass spectrometry. Nano-meter scale secondary ion mass spectrometry (NanoSIMS) combined with stable isotope labeling provides a unique tool for studying the spatiometabolic activity of microbial populations at the single cell level in order to assess both community structure and population diversity. However, assessing the distribution and range of microbial activity in complex environmental systems with slow-growing organisms, diverse carbon and nitrogen sources, or heterotrophic subpopulations poses a tremendous technical challenge because the introduction of isotopically labeled substrates frequently changes the nutrient availability and can inflate or bias measures of activity. Here, we present the use of hydrogen isotope labeling with deuterated water as an important new addition to the isotopic toolkit and apply it for the determination of single cell microbial activities by NanoSIMS imaging. This tool provides a labeling technique that minimally alters any aquatic chemical environment, can be administered with strong labels even in minimal addition (natural background is very low), is an equally universal substrate for all forms of life even in complex, carbon and nitrogen saturated systems, and can be combined with other isotopic tracers. The combination of heavy water labeling with the most commonly used NanoSIMS tracer, 15N, is technically challenging but opens up a powerful new set of multi-tracer experiments for the study of microbial activity in complex communities. We present the first truly simultaneous single cell triple isotope system measurements of 2H/1H, 13C/12C and 15N/14N and apply it to study of microbial metabolic heterogeneity and nitrogen metabolism in a continuous culture case study. Our data provide insight into both the diversity of microbial activity rates, as well as patterns of ammonium utilization at the single cell level.

Time-reversal-based SU(2)× Sn scalar invariants as (Lie Algebraic) group measures: a structured overview of generalised democratic-recoupled, uniform non-Abelian [ AX] n NMR spin systems, as abstract Sn⊃ Sn-1../U n⊃U n-1.. chain networks

NASA Astrophysics Data System (ADS)

Temme, F. P.

2004-03-01

The physics of dual group scalar invariants (SIs) as (Lie algebraic) group measures (L-GMs) and its significance to non-Abelian NMR spin systems motivates this overview of uniform general-2 n [ AX] 2 n spin evolution, which represents an extensive addendum to Corio's earlier (essentially restricted) view of Abelian spin system SU(2)-based SI-cardinalities. The |D 0( U)|((⊗SU(2)) (2n))|SI| values in [J. Magn. Reson., 134 (1998) 131] arise from strictly linear recoupled time-reversal invariance (TRI) models. In contrast, here we discuss the physical significance of an alternative polyhedral combinatorics approach to democratic recoupling (DR), a property inherent in both the TRI and statistical sampling. Recognition of spin ensemble SIs as being L-GMs over isomorphic algebras is invaluable in many DR-based NMR problems. Various [ AX] n model spin systems, including the [ AX] 3bis odd-odd parity spin system, are examined as direct applications of these L-GM- and combinatorial-based SI ideas. Hence in place of | SI|=15 (implied by Corio's | D0|((⊗ SU(2)) 2 n) approach), the bis 3-fold spin system cardinality is seen now as constrained to a single invariant on an isomorphic product algebra under L-GMs, in accord with the subspectral analysis of Jones et al. [Canad. J. Chem., 43 (1965) 683]. The group projective ideas cited here for DR (as cf. to graph theoretic views) apply to highly degenerate non-Abelian problems. Over dual tensorial bases, they define models of spin dynamical evolution whose (SR) quasiparticle superboson carrier (sub)spaces are characterised by SIs acting as explicit auxiliary labels [Physica, A198 (1993) 245; J. Math. Chem., 31 (2002) 281]. A deeper S2n network-based view of spin-alone space developed in Balasubramanian's work [J. Chem. Phys., 78 (1983) 6358] is especially important, (e.g.) in the study of spin waves [J. Math. Chem., 31 (2002) 363]. Beyond the specific NMR SIs derived here, there are DR applications where a sporadic, still higher, 2 n-fold regular uniform spin ensemble exhibits a topological FG duality to some known modest | SI| (2 i<2 n) cardinality—in principle providing for the (sparce) existence of other | SI| (2 n) DR-based values.

Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction.

PubMed Central

Dilworth, M J; Thorneley, R N

1981-01-01

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3. PMID:7030315

Bromine-80m-labeled estrogens: Auger-electron emitting, estrogen receptor-directed ligands with potential for therapy of estrogen receptor positive cancers

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeSombre, E.R.; Mease, R.C.; Hughes, A.

1988-01-01

A triphenylbromoethylene, 1,1-bis(p-hydroxyphenyl)-2-bromo-2-phenylethylene, Br-BHPE, and a bromosteroidal estrogen, 17..cap alpha..- bromovinylestradiol, BrVE/sub 2/, were labeled with the Auger electron emitting nuclide bromine-80m, prepared by the (p,n) reaction with /sup 80/Se. To assess their potential as estrogen receptor (ER) directed therapeutic substrates the bromine-80m labeled estrogens were injected into immature female rats and the tissue distribution studied at 0.5 and 2 hours. Both radiobromoestrogens showed substantial diethylstilbesterol (DES)-inhibitable localization in the ER rich tissues, uterus, pituitary, ovary and vagina at both time points. While the percent dose per gram tissue was higher for the Br-BHPE, the BrVE/sub 2/ showed higher tissuemore » to blood ratios, especially at 2 hr, reflecting the lower blood concentrations of radiobromine following administration of the steroidal bromoestrogen. Comparing intraperitoneal, intravenous and subcutaneous routes of administration for the radiobromine labeled Br-BHPE, the intraperitoneal route was particularly advantageous to provide maximum, DES-inhibitable concentrations in the peritoneal, ER-rich target organs, the uterus, ovary and vagina. While uterine concentrations after BrBHPE were from 10--48% dose/g and after BrVE/sub 2/ were 15--25% dose/g, similar treatment with /sup 80m/Br as sodium bromide showed uniform low concentrations in all tissues at about the levels seen in blood. The effective specific activity of (/sup 80m/Br)BrBHPE, assayed by specific binding to ER in rat uterine cytosol, was 8700 Ci/mmole. 23 refs., 9 figs., 2 tabs.« less

Americans and Palestinians judge spontaneous facial expressions of emotion.

PubMed

Kayyal, Mary H; Russell, James A

2013-10-01

The claim that certain emotions are universally recognized from facial expressions is based primarily on the study of expressions that were posed. The current study was of spontaneous facial expressions shown by aborigines in Papua New Guinea (Ekman, 1980); 17 faces claimed to convey one (or, in the case of blends, two) basic emotions and five faces claimed to show other universal feelings. For each face, participants rated the degree to which each of the 12 predicted emotions or feelings was conveyed. The modal choice for English-speaking Americans (n = 60), English-speaking Palestinians (n = 60), and Arabic-speaking Palestinians (n = 44) was the predicted label for only 4, 5, and 4, respectively, of the 17 faces for basic emotions, and for only 2, 2, and 2, respectively, of the 5 faces for other feelings. Observers endorsed the predicted emotion or feeling moderately often (65%, 55%, and 44%), but also denied it moderately often (35%, 45%, and 56%). They also endorsed more than one (or, for blends, two) label(s) in each face-on average, 2.3, 2.3, and 1.5 of basic emotions and 2.6, 2.2, and 1.5 of other feelings. There were both similarities and differences across culture and language, but the emotional meaning of a facial expression is not well captured by the predicted label(s) or, indeed, by any single label.

A novel method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies

NASA Astrophysics Data System (ADS)

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

2014-05-01

We developed a new method (gas-phase stripping-derivatization coupled to LC-MS) to measure the 15N atom percent excess (APE) of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye by the well-known Griess reaction in the Long Path Absorption Photometer (LOPAP). The reaction solutions containing the dye are collected at the outflow of the LOPAP, purified by solid-phase extraction and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The unlabeled azo dye (C18H19O2N5S) with a monoisotopic molecular mass of 369.41 g mol-1 can be detected as its protonated molecular ion ([M+H+], M) by HPLC-MS at a retention time of 2.8 min. Due to the natural isotope distribution M + 0, M + 1, M + 2, and M + 3 ions were considered for the calculation of the 15N APE. The optimal working range was found to be between 20 and 50% for the 15N/14N ratio. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method has been applied for the measurement of HO15NO emissions from soil in a dynamic chamber with and without spiking 15N labeled urea. Our results confirm biogenic HONO emissions from soil as HO15NO was measured after addition of 15N urea.

Kinetic 15N-isotope effects on algal growth

NASA Astrophysics Data System (ADS)

Andriukonis, Eivydas; Gorokhova, Elena

2017-03-01

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

[Injectable hospital preparation of valine labeled with the carbon 13 and nitrogen 15 (5 mg/mL) for a clinical trial on the brain tumor metabolism: Pharmaceutical control of active pharmaceutical ingredient and stability study of the finished product].

PubMed

Tall, M L; Diouf, E; Filali, S; Sauvinet, V; Laleye, D; Dhelens, C; Salmon, D; Gabert, L; Nugue, G; Sandre-Balester, C; Berger, F; Pirot, F; Pivot, C

2015-09-01

The L-Valine labeled (L-[U-(13)C,(15)N] Val) is a stable isotopic tracer administered by parenteral route within the framework of a new clinical research program concerning the brain tumor metabolism. To meet regulatory requirements and have ready to use solution with an expiration date, a pharmaceutical control of active pharmaceutical ingredient followed by stability study of hospital preparation were realised. After the pharmaceutical control of the L-[U-(13)C,(15)N] Val, the hospital preparation was prepared according to the good manufacturing preparation. Prepared bottles were stored at 5°C±3°C and 25°C±2°C for six months. The stability of the preparation was determined by physico-chemical controls (pH, osmolality, sub-visible particles, L-[U-(13)C,(15)N] Val concentration, sodium concentration, isotopic enrichment) and microbiological (bacterial endotoxin and sterility). Concentrations of L-[U-(13)C, (15)N] Val and sodium does not significantly decrease during the stability study. In parallel, no change in pH and osmolality were highlighted. Isotopic enrichment higher than 99.9% reflected the stability of labeling of L-valine molecule. The sub-visible particles, the bacterial endotoxin and sterility were in accordance with the European Pharmacopoeia attesting limpidity, apyrogenicity and sterility of this injectable preparation. The stability of this hospital preparation of L-[U-(13)C, (15)N] Val has been demonstrated for six months at 5°C±3°C and 25°C±2°C, ensuring a parenteral administration as part of the clinical trial. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

78 FR 23508 - Use of Certain Symbols in Labeling

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-19

... English language* * *'' (Sec. 801.15(c)(1)). The regulation goes on to allow for use of foreign language... 809 [Docket No. FDA-2013-N-0125] RIN 0910-AG74 Use of Certain Symbols in Labeling AGENCY: Food and... standardized symbol is part of a standard recognized by FDA for use on the labeling of medical devices (or on a...

Assessing the Consequences of Implementing Graphic Warning Labels on Cigarette Packs for Tobacco-Related Health Disparities.

PubMed

Gibson, Laura; Brennan, Emily; Momjian, Ani; Shapiro-Luft, Dina; Seitz, Holli; Cappella, Joseph N

2015-08-01

Population-level communication interventions, such as graphic warning labels (GWLs) on cigarette packs, have the potential to reduce or exacerbate tobacco-related health disparities depending on their effectiveness among disadvantaged sub-populations. This study evaluated the likely impact of nine GWLs proposed by the US Food and Drug Administration on (1) African American and (2) Hispanic smokers, who disproportionately bear the burden of tobacco-related illness, and (3) low education smokers, who have higher smoking rates. Data were collected online from current smokers randomly assigned to see GWLs (treatment) or the current text-only warning labels (control). Participants were stratified by age (18-25; 26+) in each of four groups: general population (n = 1246), African Americans (n = 1200), Hispanics (n = 1200), and low education (n = 1790). We tested the effectiveness of GWLs compared to text-only warning labels using eight outcomes that are predictive of quitting intentions or behaviors including negative emotion, intentions to hold back from smoking, intentions to engage in avoidance behaviors, and intentions to quit. Across all outcomes, GWLs were significantly more effective than text-only warning labels more often than expected by chance. Results suggested that African Americans, Hispanics and smokers with low education did not differ from the general population of smokers in their reactions to any of the nine individual GWLs. The nine GWLs were similarly effective for disadvantaged sub-populations and the general population of smokers. Implementation of GWLs is therefore unlikely to reduce or exacerbate existing tobacco-related health disparities, but will most likely uniformly increase intentions and behaviors predictive of smoking cessation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Assessing the Consequences of Implementing Graphic Warning Labels on Cigarette Packs for Tobacco-Related Health Disparities

PubMed Central

Brennan, Emily; Momjian, Ani; Shapiro-Luft, Dina; Seitz, Holli; Cappella, Joseph N.

2015-01-01

Introduction: Population-level communication interventions, such as graphic warning labels (GWLs) on cigarette packs, have the potential to reduce or exacerbate tobacco-related health disparities depending on their effectiveness among disadvantaged sub-populations. This study evaluated the likely impact of nine GWLs proposed by the US Food and Drug Administration on (1) African American and (2) Hispanic smokers, who disproportionately bear the burden of tobacco-related illness, and (3) low education smokers, who have higher smoking rates. Methods: Data were collected online from current smokers randomly assigned to see GWLs (treatment) or the current text-only warning labels (control). Participants were stratified by age (18–25; 26+) in each of four groups: general population (n = 1246), African Americans (n = 1200), Hispanics (n = 1200), and low education (n = 1790). We tested the effectiveness of GWLs compared to text-only warning labels using eight outcomes that are predictive of quitting intentions or behaviors including negative emotion, intentions to hold back from smoking, intentions to engage in avoidance behaviors, and intentions to quit. Results: Across all outcomes, GWLs were significantly more effective than text-only warning labels more often than expected by chance. Results suggested that African Americans, Hispanics and smokers with low education did not differ from the general population of smokers in their reactions to any of the nine individual GWLs. Conclusions: The nine GWLs were similarly effective for disadvantaged sub-populations and the general population of smokers. Implementation of GWLs is therefore unlikely to reduce or exacerbate existing tobacco-related health disparities, but will most likely uniformly increase intentions and behaviors predictive of smoking cessation. PMID:26180214

Single-cell measurement of archaeal and bacterial carbon assimilation in dark Pacific Ocean waters

NASA Astrophysics Data System (ADS)

Dekas, A. E.; Mayali, X.; Parada, A. E.; Fuhrman, J. A.; Weber, P. K.; Pett-Ridge, J.

2016-02-01

Microbial activity in the dark ocean plays a critical role in nutrient and elemental cycling. Here, we investigated the activity of archaea and bacteria on the single-cell level during dark incubations of Pacific Ocean water, and specifically their capacity for chemoautotrophy. Samples were collected 19 km off the coast of Los Angeles, at a depth of 150 m, and off the coast of San Francisco, at the surface. Incubations were amended with isotopically-labeled organic or inorganic carbon (13C-bicarbonate, 15N-amino acids or dual-labeled 13C-15N-amino acids), and uptake was detected using nanoscale secondary ion mass spectrometry (NanoSIMS). We analyzed 4,968 individual cells using an automated NanoSIMS analysis with particle-recognition software. After 7 days, 95% and 89% of cells (deep and shallow, respectively) demonstrated anabolic activity, i.e., incorporation of at least one isotopically-labeled substrate. Chemoautotrophy was detected at both sites, with 36% and 9% of cells (deep and shallow, respectively) assimilating 13C-bicarbonate in the dark. Fluorescence in situ hybridization coupled to NanoSIMS analysis was performed to link 16S rRNA phylogeny to patterns of C-assimilation. Thaumarchaea were found to dominate chemoautotrophy at both sites, with 13C-bicarbonate assimilation in nearly all cells hybridized with the Cren537 probe, but none hybridized with a general bacterial probe (Eub338). Conversely, widespread assimilation of both 15N and 13C from 15N-13C-amino acids was observed in the bacterial assemblage, but not in the Thaumarchaea. Interestingly, Thaumarchaeal cells were enriched in 15N after incubation with 15N-13C-amino acids, but not 13C, suggesting selective N assimilation from amino acids or substrate recycling. Together, our results demonstrate the value of single-cell measurements in characterizing patterns of C metabolism in mixed microbial community, and underscore the importance of Thaumarchaea in marine chemoautotrophy.

Synthesis of a Potent Aminopyridine-Based nNOS-Inhibitor by Two Recent No-Carrier-Added (18)F-Labelling Methods.

PubMed

Drerup, Christian; Ermert, Johannes; Coenen, Heinz H

2016-09-01

Nitric oxide (NO), an important multifunctional signaling molecule, is produced by three isoforms of NO-synthase (NOS) and has been associated with neurodegenerative disorders. Selective inhibitors of the subtypes iNOS (inducible) or nNOS (neuronal) are of great interest for decoding neurodestructive key factors, and (18)F-labelled analogues would allow investigating the NOS-function by molecular imaging with positron emission tomography. Especially, the highly selective nNOS inhibitor 6-((3-((3-fluorophenethylamino)methyl)phenoxy)methyl)-4-methylpyridin-2-amine (10) lends itself as suitable compound to be (18)F-labelled in no-carrier-added (n.c.a.) form. For preparation of the (18)F-labelled nNOS-Inhibitor [(18)F]10 a "build-up" radiosynthesis was developed based on a corresponding iodonium ylide as labelling precursor. The such activated phenethyl group of the compound was efficiently and regioselectively labelled with n.c.a. [(18)F]fluoride in 79% radiochemical yield (RCY). After conversion by reductive amination and microwave assisted displacement of the protecting groups, the desired nNOS-inhibitor was obtained in about 15% total RCY. Alternatively, for a simplified "late-stage" (18)F-labelling procedure a corresponding boronic ester precursor was synthesized and successfully used in a newer, copper(II) mediated n.c.a. (18)F-fluoro-deboroniation reaction, achieving the same total RCY. Thus, both methods proved comparatively suited to provide the highly selective NOS-inhibitor [(18)F]10 as probe for preclinical in vivo studies.

Meat Entree Item Production Guides Developed for Use in Ft. Lee Interim Central Food Preparation Facility

DTIC Science & Technology

1975-03-01

serves 8). 10. Add cooked dough . (See topping procedure). 11. Cover, label and place in blast freezer. Totals Rotes: 1. Reheating...3. Gradually add cold water and mix only enough to form a soft dough . 4. Place dough on a lightly floured board, kneeding lightly about 1...minute or until dough Is smooth. 5. Roll out to a uniform thickness of 1/4 inch. 6. Cut into 2 i/4-inch diameter biscuits. 7. Bake for 15 minutes in

Nuclear spin-lattice relaxation in nitroxide spin-label EPR.

PubMed

Marsh, Derek

2016-11-01

Nuclear relaxation is a sensitive monitor of rotational dynamics in spin-label EPR. It also contributes competing saturation transfer pathways in T 1 -exchange spectroscopy, and the determination of paramagnetic relaxation enhancement in site-directed spin labelling. A survey shows that the definition of nitrogen nuclear relaxation rate W n commonly used in the CW-EPR literature for 14 N-nitroxyl spin labels is inconsistent with that currently adopted in time-resolved EPR measurements of saturation recovery. Redefinition of the normalised 14 N spin-lattice relaxation rate, b=W n /(2W e ), preserves the expressions used for CW-EPR, whilst rendering them consistent with expressions for saturation recovery rates in pulsed EPR. Furthermore, values routinely quoted for nuclear relaxation times that are deduced from EPR spectral diffusion rates in 14 N-nitroxyl spin labels do not accord with conventional analysis of spin-lattice relaxation in this three-level system. Expressions for CW-saturation EPR with the revised definitions are summarised. Data on nitrogen nuclear spin-lattice relaxation times are compiled according to the three-level scheme for 14 N-relaxation: T 1 n =1/W n . Results are compared and contrasted with those for the two-level 15 N-nitroxide system. Copyright © 2016 Elsevier Inc. All rights reserved.

Find Pairs: The Module for Protein Quantification of the PeakQuant Software Suite

PubMed Central

Eisenacher, Martin; Kohl, Michael; Wiese, Sebastian; Hebeler, Romano; Meyer, Helmut E.

2012-01-01

Abstract Accurate quantification of proteins is one of the major tasks in current proteomics research. To address this issue, a wide range of stable isotope labeling techniques have been developed, allowing one to quantitatively study thousands of proteins by means of mass spectrometry. In this article, the FindPairs module of the PeakQuant software suite is detailed. It facilitates the automatic determination of protein abundance ratios based on the automated analysis of stable isotope-coded mass spectrometric data. Furthermore, it implements statistical methods to determine outliers due to biological as well as technical variance of proteome data obtained in replicate experiments. This provides an important means to evaluate the significance in obtained protein expression data. For demonstrating the high applicability of FindPairs, we focused on the quantitative analysis of proteome data acquired in 14N/15N labeling experiments. We further provide a comprehensive overview of the features of the FindPairs software, and compare these with existing quantification packages. The software presented here supports a wide range of proteomics applications, allowing one to quantitatively assess data derived from different stable isotope labeling approaches, such as 14N/15N labeling, SILAC, and iTRAQ. The software is publicly available at http://www.medizinisches-proteom-center.de/software and free for academic use. PMID:22909347

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

PubMed Central

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

2014-01-01

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

Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions

PubMed Central

Evans, Erin M.; Freund, Dana M.; Sondervan, Veronica M.; Cohen, Jerry D.; Hegeman, Adrian D.

2018-01-01

In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies. PMID:29904627

Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions.

PubMed

Evans, Erin M; Freund, Dana M; Sondervan, Veronica M; Cohen, Jerry D; Hegeman, Adrian D

2018-01-01

In this study we describe a [ 15 N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [ 15 N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

Metabolic patterns in Spirodela polyrhiza revealed by 15N stable isotope labeling of amino acids in photoautotrophic, heterotrophic, and mixotrophic growth conditions

NASA Astrophysics Data System (ADS)

Evans, Erin M.; Freund, Dana M.; Sondervan, Veronica M.; Cohen, Jerry D.; Hegeman, Adrian D.

2018-05-01

In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for fifteen of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of less than 100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

Conformational heterogeneity in the C-terminal zinc fingers of human MTF-1: an NMR and zinc-binding study.

PubMed

Giedroc, D P; Chen, X; Pennella, M A; LiWang, A C

2001-11-09

The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys(2)-His(2) motifs, each of which was projected to form well-structured betabetaalpha domains upon Zn(II) binding. In this report, the structure and backbone dynamics of a fragment containing the unusual C-terminal fingers F4-F6 has been investigated. (15)N heteronuclear single quantum coherence (HSQC) spectra of uniformly (15)N-labeled hMTF-zf46 show that Zn(II) induces the folding of hMTF-zf46. Analysis of the secondary structure of Zn(3) hMTF-zf46 determined by (13)Calpha chemical shift indexing and the magnitude of (3)J(Halpha-HN) clearly reveal that zinc fingers F4 and F6 adopt typical betabetaalpha structures. An analysis of the heteronuclear backbone (15)N relaxation dynamics behavior is consistent with this picture and further reveals independent tumbling of the finger domains in solution. Titration of apo-MTF-zf46 with Zn(II) reveals that the F4 domain binds Zn(II) significantly more tightly than do the other two finger domains. In contrast to fingers F4 and F6, the betabetaalpha fold of finger F5 is unstable and only partially populated at substoichiometric Zn(II); a slight molar excess of zinc results in severe conformational exchange broadening of all F5 NH cross-peaks. Finally, although Cd(II) binds to apo-hMTF-zf46 as revealed by intense S(-)-->Cd(II) absorption, a non-native structure results; addition of stoichiometric Zn(II) to the Cd(II) complex results in quantitative refolding of the betabetaalpha structure in F4 and F6. The functional implications of these results are discussed.

High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy

NASA Astrophysics Data System (ADS)

Jaroniec, Christopher P.; Macphee, Cait E.; Bajaj, Vikram S.; McMahon, Michael T.; Dobson, Christopher M.; Griffin, Robert G.

2004-01-01

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete 13C and 15N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 13C-15N distances between 3 and 6 Å were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly 13C, 15N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended -strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

Clipping and shading alter NH4+ uptake by plants in grazed and ungrazed Tibetan alpine grasslands

NASA Astrophysics Data System (ADS)

Sun, Yue; Schleuss, Per; Li, Qianru; Yang, Baijie; Xu, Xingliang; Kuzyakov, Yakov

2014-05-01

The Kobresia pastures are the most common and most important vegetation type on the Tibetan Plateau as it occupies more than 35% the plateau area. These pastures have been remained stable for about one million years, but have been strongly changed by increased grazing in the recent decades which led to serious grassland degradation. Previous studies on the N cycling in alpine grasslands showed that plant growth was limited by low N availability due to low N mineralization caused by low temperature. However, the effect of grazing on N turnover processes and plant N uptake remains unclear. To clarify the grazing effect for a better understanding N mineralization and plant N uptake in these alpine grasslands, we conducted a 15N experiment in grazed and ungraded plots in these alpine grasslands. Because ammonium was a dominant N form, we used 15N-labeled ammonium so that we can also measure gross N mineralization. To explore the effect of root exudates on 15NH4+ uptake by plants and gross N mineralization, three treatments such as clipping, shading and control were used. Initially, all treatments were labeled by 15NH4+, with blank treatments no 15N tracer addition. Plant and soil samples were collected 7, 14 and 28 days after the labelling. 15NH4+ uptake by alpine plants almost did not change after clipping in the grazed plots, but its uptake was lower under the clipping treatment than under the control treatment in the ungrazed plots. 15N recovery in plants under the shading treatment remained the lowest level in grazed and ungrazed plots. Although clipping removed a part of aboveground biomass, subsequent stimulation of plant growth increased N uptake by plants. Likely, moderate grazing removed a part of aboveground biomass, but 15N recovery in plants was still compared to that in the ungrazed plots, indicating moderate grazing stimulate N uptake by plants through compensatory growth. Gross N mineralization under the shading treatment was higher than under the clipping treatment (shading vs clipping: 0.42 vs 0.34 mg N kg-1 h-1) in the grazed plot. In contrast, gross N mineralization was lower for shading treatment than for clipping treatment (shading vs clipping 0.47 vs 0.63 mg N kg-1 h-1) in the ungrazed plot. Gross N mineralization in the ungrazed soil was higher than in the grazed soil, suggesting that grazing greatly reduced the potential to provide available nitrogen for plants and microorganisms. Therefore, we concluded that low photosynthesis caused by shading, clipping and grazing can affect N transformation and therefore affect the format of soil organic matter.

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies.

PubMed

Gande, Santosh L; Saxena, Krishna; Sreeramulu, Sridhar; Linhard, Verena; Kudlinzki, Denis; Heinzlmeir, Stephanie; Reichert, Andreas J; Skerra, Arne; Kuster, Bernhard; Schwalbe, Harald

2016-12-02

The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells). 1 H, 15 N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596-G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2-ligand crystal structures. We have also established a strategy for selective and uniform 15 N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2-drug interactions by NMR. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solid-state NMR characterization of copolymers of nylon 11 and nylon 12.

PubMed

Johnson, C G; Mathias, L J

1997-05-01

Solid-state 13C and 15N NMR spectroscopy, in conjunction with differential scanning calorimetry, wide-angle X-ray diffraction and infrared spectroscopy, were used to characterize a series of nylon 11 and 12 copolymers with mole percentages of nylon 12 monomer of 0, 15, 35, 50, 65, 85, and 100%. Monotonic melting point (Tm) and heat of fusion depressions were observed for the copolymer series with the 65 mol% nylon 12 copolymer having the lowest apparent crystallinity and Tm at 148 degrees C. Solid-state 15N NMR spectra showed a smooth shift of the main peak position for the as-prepared copolymers from 84 ppm for the alpha-form of pure nylon 11 to 89 ppm for the gamma-form of pure nylon 12. Similar behavior was seen for FTIR amide V and VI modes which are also sensitive to the alpha- and gamma-crystal forms. 13C NMR T1 measurements showed that the overall most mobile sample was the 65:35 copolymer. The amide group of the 1:1 copolymer was labelled using 15N-labelled amino acids available through the Gabriel synthesis; an annealed, solution-cast film of this sample showed a T1N value of 349 s, similar to values seen for annealed nylon 11 and nylon 12 homopolymers. The WAXS pattern for the 65 mol% nylon 12 sample showed a sharp peak at 2 theta = 21.3, overlapping a broad peak centered at 2 theta = 21.0. These are consistent with the values seen for gamma-form nylon 12. The 1:1 copolymer (15N labelled) was shown to be polymorphic, like the homopolymers after specific treatments, with a gamma-like phase formed upon solvent casting, and an alpha-like phase dominating for as-polymerized material and precipitated flakes.

Effect of different forage species on the nitrogen uptake in Hulunbeir

NASA Astrophysics Data System (ADS)

Xu, Li-Jun; Xu, Xing-Liang; Tang, Xue-Juan; Yang, Gui-Xia; Zhang, Zhao; Xin, Xiao-Ping

2017-02-01

Knowledge of determining factors for nitrogen uptake preferences and how they are modified in changing environments are critical to understand ecosystem nitrogen cycling and to predict plant responses to future environmental changes. However, it remains unclear in this aspect for the main managed grassland (Medicago sativa, Bromus inermis, Leymus chinensis) and crop (Brassica campestris) under field condition in Hulunbeir area of Inner Mongolia of China. Two 15N tracer experiments utilizing a unique differential labelled nitrogen source were employed in both managed grassland (M. sativa, B. inermis and L. chinensis) and crop (B. campestris) in Hulunbeir area of Inner Mongolia of China. Tracing both labels in the above-and below ground plant biomass, soil NH4 + -N or NO3 - -N and NH4 + -N or NO3 - -N uptake by plants. There were differences between soil NO3 --N concentration and NH4+-N concentration, and NO3 --N concentration was higher than NH4 +-N concentration. NO3 --N concentration was led by different grass species. The NH4 +-N concentration in August were higher than in July on the whole, the highest value for B. campestris and the lowest for B. inermis. The plant N concentration in B. inermis, L. chinensis and B. campestris showed decreasing trend, its mean value decreased by 20.1, 47.9 and 26.7%, respectively, and M. sativa increased by 13.7%. Among the four species, the individuals exhibited a preference for 15NO3 -, indicated by higher 15N signatures in 15NO3-treatment than in 15NH4 + treatment.

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

NASA Astrophysics Data System (ADS)

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

2015-04-01

Denitrification, the reduction of oxidized forms of inorganic N to N2O and N2 is an important pathway of gaseous nitrogen losses. Measuring denitrification, especially the reduction of N2O to N2, expressed in the product ratio (N2O/(N2O + N2)), is rather difficult and hence rarely performed under field conditions. But using the 15N gas-flux method allows determining N transformation processes in their natural environment. In order to develop effective climate mitigation strategies understanding the N2O source is essential. We used the 15N gas-flux method to determine N2O and N2 emissions following grassland renewal and conversion techniques. Therefore we selected three different treatments: control (C), mechanical grassland renovation (GR) (autumn 2013) and grassland conversion to maize (GM) (spring 2014) from field plot trials on two different sites (Histic Gleysoil and Plaggic Anthrosol) near Oldenburg, Lower Saxony, Germany. We applied 15N labeled KNO3- (60 atom. % 15N) at a rate equivalent to common farming practices (150 kg N*ha-1) using needle injection of fertilizer solution in three different depths (10 cm, 15 cm, 20 cm) for homogeneous soil labeling up to 30 cm in microplots. During the first 10 days after application (May 2014) gas flux measurements from closed chambers were performed every second day and then weekly following a period of 8 weeks. Gas samples were analyzed for δ15N of N2 and N2O by IRMS according to Lewicka-Szczebak et al. (2013). Concentration and 15N enrichment of NO3- in soil water was determined on weekly samples using the SPIN-MAS technique (Stange et al. 2007). Fluxes of N2 and N2O evolved from the 15N labeled soil nitrogen pool were calculated using the equations of Spott et al. (2006). Peak events of N2 and N2O emissions occurred during the first 10 days of measurement, showing differences in soil types, as well as treatment variations. N2 fluxes up to 178 g*ha-1*day-1 and N2O fluxes up to 280 g*ha-1*day-1 were measured on the Plaggic Anthrosol in the GR treatment, while on the Histic Gleysoil, the GM treatment showed highest fluxes with N2 fluxes up to 1260 g*ha-1*day-1 and N2O fluxes up to 747 g*ha-1*day-1. Alike the product ratio of initial fluxes was higher on the Plaggic Anthrosol and lower on the Histic Gleysoil. Data analysis is still in progress and further results will be provided. References: Lewicka-Szczebak, D., R. Well, A. Giesemann, L. Rohe and U. Wolf (2013). "An enhanced technique for automated determination of 15N signatures of N2, (N2+N2O) and N2O in gas samples." Rapid Communications in Mass Spectrometry 27(13): 1548-1558. Spott, O., R. Russow, B. Apelt and C. F. Stange (2006). "A 15N-aided artificial atmosphere gas flow technique for online determination of soil N2 release using the zeolite Köstrolith SX6®." Rapid Communications in Mass Spectrometry 20(22): 3267-3274. Stange, F., O. Spott, B. Apelt and R. W. Russow (2007). "Automated and rapid online determination of 15N abundance and concentration of ammonium, nitrite, or nitrate in aqueous samples by the SPINMAS technique." Isotopes in Environmental and Health Studies 43(3): 227-236.

Distribution and recovery of nitrogen-15-labeled liquid anhydrous ammonia among various soil fractions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Norman, R.J.; Kurtz, L.T.; Stevenson, F.J.

Since liquid anhydrous ammonia (LAA) is a major N fertilizer, information was sought about the proportions of LAA that enter into various combinations in soils. Liquid anhydrous NH/sub 3/, labeled with /sup 15/N was injected into three soils (Drummer, Typic Haplaquoll; Blount, Aeric Ochraqualf; Cisne, Mollic Albaqualf) in the laboratory at a rate equivalent to a field application of 206 kg N ha /sup 1/ in 76.2 cm knife-spacings. At 1, 7, 14, 28, 56, and 112 d after application, fertilizer N present in different soil fractions was determined in five concentric zones with radii of 0 to 1.5, 1.5more » to 3.0, 3.0 to 4.5, 4.5 to 6.0, and 6.0 to 7.0 cm around the point of application. Depending on the soil, from 68 to 83% of the applied /sup 15/N was accounted for as (exchangeable NH/sub 4//sup +/ + NO/sub 3//sup -/ + NO/sub 2//sup -/)-N by the 112th day following application, the remainder being accounted for as clay-fixed NH/sub 4//sup +/ (1.9-4.9%), organic matter-fixed NH/sub 3/ (4.0-6.0%), and biologically immobilized organic N (3.9-9.3%). From 50 to 70% of the organic matter-fixed NH/sub 3/-N was released by hydrolysis with dilute KOH solution as compared to 10 to 15% for the immobilized N. Total recovery of /sup 15/N at 112 d ranged from 77% for the Cisne soil to about 97% for the Drummer and Blount soils. Lateral distributions and transformations of NH/sub 4//sup +/ and NO/sub 3//sup -/ and pH trends after LAA applications were similar to those reported by previous investigators.« less

Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

PubMed

Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

2010-01-01

The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

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

PubMed Central

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

2011-01-01

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

Investigating Microbial Activity in Diazotrophic Methane Seep Sediment via Transcript Analysis and Single-Cell FISH-NanoSIMS

NASA Astrophysics Data System (ADS)

Dekas, A. E.; Connon, S. A.; Chadwick, G.; Orphan, V. J.

2012-12-01

Methane seep microbial ecosystems are phylogenetically diverse and physiologically complex, and require culture-independent techniques to accurately investigate metabolic activity. In the present study we combine an RNA analysis of four key microbial genes with FISH-NanoSIMS analysis of single cells to determine the diversity of nitrogen fixing microorganisms (diazotrophs) present at a deep-sea methane-seeping site, as well as investigate the methane-dependency of a variety of community members. Recently, methane-dependent nitrogen fixation was observed in Mound 12 Costa Rica sediments, and was spatially correlated with the abundance of aggregates of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacterial symbionts (SRB). Combined with the detection of 15N uptake from 15N2 in these aggregates, this suggested that the ANME-SRB aggregates are the primary diazotrophs in seep sediment. However, the diversity of dinitrogenase reductase (nifH) sequences recovered from several deep-sea locales, including Mound 12, suggests a greater diversity of diazotrophs in marine sediment. To investigate the activity of these potential diazotrophs in Mound 12 sediment, we investigated a suite of RNA transcripts in 15N2 incubations in both the presence and absence of methane: nifH, bacterial 16S rRNA, methyl coenzyme M reductase A (mcrA), and adenosine-5'-phosposulfate reductase alpha subunit (aprA). No nifH transcripts were recovered in incubations without methane, consistent with previous measurements lacking 15N2 uptake in the same sediments. The activity of the bacterial community in general, assessed by variable transcription, was also greatly affected by the presence or absence of methane. Single-cell fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS) was employed to confirm diazotrophic activity (15N2 uptake) and protein synthesis (15NH4+ uptake) of particular species implicated as ecologically important by the pattern of transcripts recovered in the mesocosm experiments. This analysis revealed 15N enrichment in free-living (i.e. non-ANME associated) members of the Desulfobulbaceae in 15N2 incubations with methane, while free-living Desulfosarcina/Desulfococcus cells, as well as nearly 40 unidentified DAPI-stained cells, were not 15N enriched. However, further NanoSIMS analyses of DSB in a variety of incubation conditions suggests that this enrichment may be due to N sharing between the ANME and DSB while in tight physical association, and then subsequent dissociation, rather than nitrogen fixation by the DSB. If true, this is an excellent example of the potential pitfalls of single cell stable isotope labeling experiments, and potential false positives due to the recycling of labeled material between (even transiently) closely associated symbionts. This work highlights both the utility of transcript analysis as a hypothesis-generator for direct analyses of microbial activity via stable isotope labeling, as well as the need to contextualize labeling experiments with investigations of microbial community structure.

Caffeine Content in Popular Energy Drinks and Energy Shots.

PubMed

Attipoe, Selasi; Leggit, Jeffrey; Deuster, Patricia A

2016-09-01

The use of energy beverages is high among the general population and military personnel. Previous studies have reported discrepancies between the actual amount of caffeine in products and the amount of caffeine on stated labels. Thus, the purpose of this study was to examine the content of caffeine listed on the labels of various energy drinks and energy shots. Top-selling energy drinks (n = 9) and energy shots (n = 5) were purchased from retail stores. Three of each of the 14 products were purchased and analyzed for caffeine content by an independent laboratory. Of the 14 products tested, 5 did not provide caffeine amounts on their facts panel-of those, 3 listed caffeine as an ingredient and 2 listed caffeine as part of a proprietary blend. The remaining 9 (of 14) products stated the amounts of caffeine on their labels, all of which were within 15% of the amount indicated on the label. In this study, although the energy beverages that indicated the amount of caffeine it contained had values within ±15% of the amount listed on the label, a potentially acceptable range, this finding is not acceptable with regard to current labeling regulations, which require added ingredients to total 100%. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

Use of Nitrogen-15-Enriched Escherichia coli as a Bacterial Tracer in Karst Aquifers.

PubMed

Ward, James W; Warden, John G; Bandy, Ashley M; Fryar, Alan E; Brion, Gail M; Macko, Stephen A; Romanek, Christopher S; Coyne, Mark S

2016-11-01

Karst aquifers are susceptible to contamination by microorganisms, but relatively few studies have used bacteria as tracers. We demonstrate the utility of Escherichia coli enriched in the stable isotope nitrogen-15 ( 15 N) as a novel bacterial tracer. Nonpathogenic E. coli from two springs in central Kentucky were grown on 15 N-enriched media. Survival of E. coli and persistence of the isotopic signal were assessed in two sets of laboratory experiments conducted with sterilized spring water in dark microcosms at 14 °C. First, isotopically labeled bacteria survived for 130 d at concentrations within one log unit of the average initial value, and there was no significant difference in δ 15 N values from Day 1 to Day 130. Second, water samples with E. coli were inoculated with either of two different species of protozoa (Tetrahymena pyriformis or Colpoda steinii). During 7 d, δ 15 N values increased in T. pyriformis while bacterial populations decreased. In a field test, following a 2.1-cm rainfall, 15 N-labeled E. coli, solutes (rhodamine WT dye and bromide), and latex microspheres were injected into a sinkhole approximately 530 m upgradient of a spring. Breakthrough of all tracers coincided, but microspheres were remobilized by subsequent storms, unlike other tracers. Enriched E. coli exhibited more tailing than solute tracers during the initial storm-flow recession. These results indicate that 15 N-enriched E. coli is a viable tracer of bacterial transport in karst aquifers, although predation may attenuate the isotopic signal in systems that are not rapidly flushed. © 2016, National Ground Water Association.

Advanced oxidation chemistry of paracetamol. UV/H(2)O(2)-induced hydroxylation/degradation pathways and (15)N-aided inventory of nitrogenous breakdown products.

PubMed

Vogna, Davide; Marotta, Raffaele; Napolitano, Alessandra; D'Ischia, Marco

2002-08-23

The advanced oxidation chemistry of the antipyretic drug paracetamol (1) with the UV/H(2)O(2) system was investigated by an integrated methodology based on (15)N-labeling and GC-MS, HPLC, and 2D (1)H, (13)C, and (15)N NMR analysis. Main degradation pathways derived from three hydroxylation steps, leading to 1,4-hydroquinone/1,4-benzoquinone, 4-acetylaminocatechol and, to a much lesser extent, 4-acetylaminoresorcine. Oxidation of the primary aromatic intermediates, viz. 4-acetylaminocatechol, 1,4-hydroquinone, 1,4-benzoquinone, and 1,2,4-benzenetriol, resulted in a series of nitrogenous and non-nitrogenous degradation products. The former included N-acetylglyoxylamide, acetylaminomalonic acid, acetylaminohydroxymalonic acid, acetylaminomaleic acid, diastereoisomeric 2-acetylamino-3-hydroxybutanedioic acids, 2-acetylaminobutenedioic acid, 3-acetylamino-4-hydroxy-2-pentenedioic acid, and 2,4-dihydroxy-3-acetylamino-2-pentenedioic acid, as well as two muconic and hydroxymuconic acid derivatives. (15)N NMR spectra revealed the accumulation since the early stages of substantial amounts of acetamide and oxalic acid monoamide. These results provide the first insight into the advanced oxidation chemistry of a 4-aminophenol derivative by the UV/H(2)O(2) system, and highlight the investigative potential of integrated GC-MS/NMR methodologies based on (15)N-labeling to track degradation pathways of nitrogenous species.

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

PubMed

Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

2016-10-01

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

Nicotine demethylation in Nicotiana cell suspension cultures: N'-formylnornicotine is not involved.

PubMed

Bartholomeusz, Trixie Ann; Bhogal, Ramneek K; Molinié, Roland; Felpin, François-Xavier; Mathé-Allainmat, Monique; Meier, Anna-Carolin; Dräger, Birgit; Lebreton, Jacques; Roscher, Albrecht; Robins, Richard J; Mesnard, François

2005-10-01

Nicotine or nornicotine enriched with stable isotopes in either the N'-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography-mass spectroscopy (GC-MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N'-demethylation product. In addition, six minor metabolites appeared during the course of nicotine metabolism, four of which were identified as cotinine, myosmine, N'-formylnornicotine and N'-carboethoxynornicotine. While cotinine was formed from [(13)C,(2)H(3)-methyl]nicotine without dilution of label, N'-formylnornicotine was labelled at only about 6% of the level of nicotine and N'-carboethoxynornicotine was unlabelled. Feeding with [1'-(15)N]nornicotine resulted in incorporation without dilution of label into both N'-formylnornicotine and N'-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N'-formylnornicotine and N'-carboethoxynornicotine are metabolites of nornicotine. Thus, it is directly demonstrated that N'-formylnornicotine is not an intermediate in nicotine demethylation.

Three Drug Combinations for Late-Stage Trypanosoma brucei gambiense Sleeping Sickness: A Randomized Clinical Trial in Uganda

PubMed Central

Priotto, Gerardo; Fogg, Carole; Balasegaram, Manica; Erphas, Olema; Louga, Albino; Checchi, Francesco; Ghabri, Salah; Piola, Patrice

2006-01-01

Objectives: Our objective was to compare the efficacy and safety of three drug combinations for the treatment of late-stage human African trypanosomiasis caused by Trypanosoma brucei gambiense. Design: This trial was a randomized, open-label, active control, parallel clinical trial comparing three arms. Setting: The study took place at the Sleeping Sickness Treatment Center run by Médecins Sans Frontières at Omugo, Arua District, Uganda Participants: Stage 2 patients diagnosed in Northern Uganda were screened for inclusion and a total of 54 selected. Interventions: Three drug combinations were given to randomly assigned patients: melarsoprol-nifurtimox (M+N), melarsoprol-eflornithine (M+E), and nifurtimox-eflornithine (N+E). Dosages were uniform: intravenous (IV) melarsoprol 1.8 mg/kg/d, daily for 10 d; IV eflornithine 400 mg/kg/d, every 6 h for 7 d; oral nifurtimox 15 (adults) or 20 (children <15 y) mg/kg/d, every 8 h for 10 d. Patients were followed up for 24 mo. Outcome Measures: Outcomes were cure rates and adverse events attributable to treatment. Results: Randomization was performed on 54 patients before enrollment was suspended due to unacceptable toxicity in one of the three arms. Cure rates obtained with the intention to treat analysis were M+N 44.4%, M+E 78.9%, and N+E 94.1%, and were significantly higher with N+E (p = 0.003) and M+E (p = 0.045) than with M+N. Adverse events were less frequent and less severe with N+E, resulting in fewer treatment interruptions and no fatalities. Four patients died who were taking melarsoprol-nifurtimox and one who was taking melarsoprol-eflornithine. Conclusions: The N+E combination appears to be a promising first-line therapy that may improve treatment of sleeping sickness, although the results from this interrupted study do not permit conclusive interpretations. Larger studies are needed to continue the evaluation of this drug combination in the treatment of T. b. gambiense sleeping sickness. PMID:17160135

DOES NITROGEN PARTITIONING PROMOTE SPECIES DIVERSITY IN ARCTIC TUSSOCK TUNDRA?

EPA Science Inventory

We used 15N soil-labeling techniques to examine how the dominant species in a N-limited, tussock tundra plant community partitioned soil N, and how such partitioning may contribute to community organization. The five most productive species were well differentiated with respect ...

Sunburn protection as a function of sunscreen application thickness differs between high and low SPFs.

PubMed

Liu, Wei; Wang, Xuemin; Lai, Wei; Yan, Tian; Wu, Yanyu; Wan, Miaojian; Yi, Jinling; Matsui, Mary S

2012-06-01

Sunscreens are an important component of healthy sun-protection behavior. To achieve satisfactory protection, sunscreens must be applied consistently, evenly and correctly. Consumers do not apply sunscreen properly and, therefore, do not achieve the protection indicated by the label 'sun protection factor' (SPF). The objective of the present study was to determine the actual sun(burn) protection given by a range of sunscreen application thickness levels for both low and high SPF formulas. Forty study subjects were recruited from each of three geographical regions in China. Sunscreens with label SPFs of 4, 15, 30, and 55 were tested at application levels of 0.5, 1.0, 1.5, and 2.0 mg/cm(2) in three laboratories using a standard SPF protocol. Sunscreens with lower SPFs (4 and 15) showed a linear dose-response relationship with application level, but higher SPF (30 and 55) product protection was exponentially related to application thickness. Sunscreen protection is not related in one uniform way to the amount of product applied to human skin. Consumers may achieve an even lower than expected sunburn protection from high SPF products than from low SPF sunscreens. © 2012 John Wiley & Sons A/S.

Production of antibody labeled gold nanoparticles for influenza virus H5N1 diagnosis kit development

NASA Astrophysics Data System (ADS)

Pham, Van Dong; Hoang, Ha; Hoang Phan, Trong; Conrad, Udo; Chu, Hoang Ha

2012-12-01

Preparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its use in developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles (AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1 (scFv7 and scFv24) were produced using pTI2 + vector and E. coli strain HB2151. These antibodies were purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The method for preparation of antibody-colloidal gold conjugate was based on electrostatic force binding antibody with colloidal gold. The effect of factors such as pH and concentration of antibody has been quantitatively analyzed using spectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. The morphological study by scanning electron microscopy (SEM) showed that the average size of the spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidal AuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum. The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity of synthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blot immunological method. The results confirmed the ability in detection of the A/H5N1 virus of the prepared antibody labeled gold particles and opened up the possibility of using them in manufacturing rapid detection kit for this virus.

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part I. Chemistry and labeling technique.

PubMed

Schuhmacher, J; Matys, R; Hauser, H; Maier-Borst, W; Matzku, S

1986-01-01

As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N,N'-di-[(o-hydroxyphenyl) acetic acid] (P-EDDHA), which tightly complexes 67Ga, was synthesized. The 67Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied 67Ga. In vitro stability was evaluated in human serum at 37 degrees C and showed a half-life of about 120 h for the release of 67Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for 111In-DTPA labeled Abs. Because of the high stability of the 67Ga-P-EDDHA chelate, the in vivo formation of radioactive labeled transferrin by transchelation, as described for 111In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

Environmental and Individual Factors Affecting Menu Labeling Utilization: A Qualitative Research Study

PubMed Central

Schindler, Jennifer; Kiszko, Kamila; Abrams, Courtney; Islam, Nadia; Elbel, Brian

2013-01-01

Obesity is a significant public health concern that disproportionally affects low-income and minority populations. Recent policies mandating the posting of calories on menus in fast food chain restaurants have not proven to uniformly influence food choice. This qualitative research study uses focus groups to study individual and environmental factors affecting the usage of these menu labels among low-income, minority populations. Ten focus groups targeting low-income residents (n=105) were conducted at various community organizations throughout NYC in Spanish, English, or a combination of both languages, over a nine-month period in 2011. In late 2011 and early 2012, transcripts were coded through the process of thematic analysis using Atlas.ti for naturally emerging themes, influences, and determinants of food choice. Few used menu labels, despite awareness. Among the themes pertaining to menu label usage, price and time constraints, confusion and lack of understanding of caloric values, as well as the priority of preference, hunger, and habitual ordering habits were most frequently cited as barriers to menu label usage. Based on the individual and external influences on food choice that often take priority over calorie consideration, a modified approach may be necessary to make menu labels more effective and user-friendly. PMID:23402695

Pleomorphic copper coordination by Alzheimer's disease amyloid-beta peptide.

PubMed

Drew, Simon C; Noble, Christopher J; Masters, Colin L; Hanson, Graeme R; Barnham, Kevin J

2009-01-28

Numerous conflicting models have been proposed regarding the nature of the Cu(2+) coordination environment of the amyloid beta (Abeta) peptide, the causative agent of Alzheimer's disease. This study used multifrequency CW-EPR spectroscopy to directly resolve the superhyperfine interactions between Cu(2+) and the ligand nuclei of Abeta, thereby avoiding ambiguities associated with introducing point mutations. Using a library of Abeta16 analogues with site-specific (15)N-labeling at Asp1, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu(2+) coordination modes, {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H13)} (component Ia) and {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H14)} (component Ib), between pH 6-7. A third coordination mode (component II) was identified at pH 8.0, and simulation of the superhyperfine resonances indicated a 3N1O coordination sphere involving nitrogen ligation by His6, His13, and His14. No differences were observed upon (17)O-labeling of the phenolic oxygen of Tyr10, confirming it is not a key oxygen ligand in the physiological pH range. Hyperfine sublevel correlation (HYSCORE) spectroscopy, in conjunction with site-specific (15)N-labeling, provided additional support for the common role of His6 in components Ia and Ib, and for the assignment of a {O, N(epsilon)(H6), N(epsilon)(H13), N(epsilon)(H14)} coordination sphere to component II. HYSCORE studies of a peptide analogue with selective (13)C-labeling of Asp1 revealed (13)C cross-peaks characteristic of equatorial coordination by the carboxylate oxygen of Asp1 in component Ia/b coordination. The direct resolution of Cu(2+) ligand interactions, together with the key finding that component I is composed of two distinct coordination modes, provides valuable insight into a range of conflicting ligand assignments and highlights the complexity of Cu(2+)/Abeta interactions.

The effect of elevated atmospheric CO2 concentration on gross nitrogen and carbon dynamics in a permanent grassland: A field pulse-labeling study

NASA Astrophysics Data System (ADS)

Moser, Gerald; Gorenflo, André; Keidel, Lisa; Brenzinger, Kristof; Elias, Dafydd; McNamara, Niall; Maček, Irena; Vodnik, Dominik; Braker, Gesche; Schimmelpfennig, Sonja; Gerstner, Judith; Müller, Christoph

2014-05-01

To predict ecosystem reactions to elevated atmospheric CO2 (eCO2) it is essential to understand the interactions between plant carbon input, microbial community composition and activity and associated nutrient dynamics. Long-term observations (> 14 years) within the Giessen Free Air Carbon dioxide Enrichment (Giessen FACE) study on permanent grassland showed next to an enhanced biomass production an unexpected strong positive feedback effect on ecosystem respiration and nitrous oxide (N2O) production. The overall goal of this study is to understand the long-term effects of eCO2 and carbon input on microbial community composition and activity as well as the associated nitrogen dynamics, N2O production and plant N uptake in the Giessen FACE study on permanent grassland. A combination of 13CO2 pulse labelling with 15N tracing of 15NH4+ and 15NO3- was carried out in situ. Different fractions of soil organic matter (recalcitrant, labile SOM) and the various mineral N pools in the soil (NH4+, NO3-), gross N transformation rates, pool size dependent N2O and N2 emissions as well as N species dependent plant N uptake rates and the origin of the CO2 respiration have been quantified. Microbial analyses include exploring changes in the composition of microbial communities involved in the turnover of NH4+, NO3-, N2O and N2, i.e. ammonia oxidizing, denitrifying, and microbial communities involved in dissimilatory nitrate reduction to ammonia (DNRA). mRNA based analyses will be employed to comparably evaluate the long-term effects of eCO2 on the structure and abundance of these communities, while transcripts of these genes will be used to target the fractions of the communities which actively contribute to N transformations. We quantified the contribution of mycorrhizae on N2O emissions and observed the phenological development of the mycorrhizae after the labeling.

Enhanced plant growth at reduced N2O emissions: 15N dynamics confirm nitrate capture and release of co-composted biochar

NASA Astrophysics Data System (ADS)

Kammann, Claudia; Schmidt, Hans-Peter; Williams, Anne; Hagemann, Nikolas; Marhan, Sven; Clough, Tim; Mueller, Christoph

2017-04-01

Pyrogenic carbon (biochar) produced from biomass may be used as a soil amendment to sequester biomass-C and to mitigate climate change. Moreover, biochar may increase soil fertility and optimize nutrient cycling in agro-ecosystems. However, according to meta-studies large additions (>10 t ha-1) of pure biochar, may only lead to moderate yield increases. Thus, there are no economic benefits for farmers to use biochar. Recently, it has been suggested that biochar may be used as an on-farm nutrient management tool (feed additive, liquid manure treatment, composting) to deliver small doses of nutrients and biochar to the soil each year. It may also be used as a carrier for (organic) nutrients in small doses (0.5 - 2 t ha-1) in the root zone; recent studies reported considerably increased yields. The mechanisms, however, are not well understood. Co-composted biochar was recently shown to promote plant growth due to its nutrient delivery and release capabilities, particularly nitrate (NO3-). To gain further insights into biochar-nitrogen (N) interactions, we conducted a 15N labelling-tracing study under controlled conditions with treatments consisting of a non-biochar amended control, 2% (wt/wt) of untreated biochar particles (BCpure ,no intrinsic content of nitrate) and a 2% co-composted biochar (BCcomp, 5.3 g NO3-N kg-1 as a result of composting), replicated thrice. Biochars were added to a sandy loam soil in jars (200 g) planted with barley (Hordeum vulgare L.) seedlings. Fertilizer (NH4NO3) was homogenously added with either the ammonium (NH4+) or the nitrate (NO3-) pools 15N labelled (60 atom% 15N) hours before planting the seedlings. Sets of 18 jars were harvested on days 1, 3, 8, 15 and 29 and nitrogen pools were analysed to trace 15N fertilizer fate (soil mineral N, plant biomass, biochar particles, nitrous oxide (N2O) emissions). Interestingly, both BCpure and BCcomp captured fertilizer 15N from the soil matrix within hours of addition, with higher capture of 15N-NO3 than 15N-NH4. Surprisingly, the already nitrate-enriched BCcomp captured significantly more 15N than the non-enriched BCpure. Plant biomass increased significantly in the BCcomp compared to both BCpure and control treatment, because BCcomp still delivered N to the growing plants when soil mineral N was already depleted. N2O emissions were lower in the BCcomp treatment (by 60%) and the BCpure treatment (by 67%) compared to the control, despite higher levels of mineral N introduced with BCcomp. The 15N-N2O enrichment suggested that with biochar a relatively larger proportion was contributed by nitrification (although denitrification still dominated); and that accelerated mineralization-nitrification from unlabelled soil organic N pools diluted the 15N-nitrate label. Our results suggest that nitrate capture in biochar particles may be one of the mechanism among others reducing N2O emissions. In conclusion, modifying and designing biochars by organic pre-treatments may promote plant growth while N2O emission reduction is still possible. However more research is needed to achieve the envisaged economic and environmental benefits of C-sequestering biochar use in agriculture. Acknowledgement: CK gratefully acknowledges the financial support of DFG grant no. Ka3442/1-1.

Uptake of stormwater nitrogen in bioretention systems demonstrated from 15N tracer techniques

NASA Astrophysics Data System (ADS)

Houdeshel, D.; Hultine, K. R.; Pomeroy, C. A.

2012-12-01

Bioretention stormwater management systems are engineered ecosystems that capture urban stormwater in order to reduce the harmful effects of stormwater pollution on receiving waters. Bioretention systems have been shown to be effective at reducing the volume of runoff, and thereby reduce the nutrient loading to receiving waters from urban areas. However, little work has been done to evaluate the treatment processes that are responsible for reductions in effluent nitrogen (N). We hypothesize that the pulses of inorganic nitrogen associated with urban runoff events are captured in the plat tissues within these systems and not adsorbed to the soil media, thus creating a long-term, sustainable treatment approach to reducing the total nutrient loading to receiving waters. Nitrogen treatment performance was tested on two bioretention systems in Salt Lake City, UT: 1) an upland native community that does not require irrigation in semi-arid climates, and 2) a wetland community that requires 250 l of daily irrigation to offset the relatively high evaporative demand in the region. Each cell is sized to treat a 2.5 cm storm from a 140 m2 impervious surface: the area of the bioretention system is 10 m2. To test the N removal performance of each system, runoff events were simulated to represent an average precipitation regime using a synthetic stormwater blend starting in January, 2012. Effluent was collected from an underdrain and analyzed for total nitrogen (TN); mass removal was calculated for each month by subtracting the TN mass added to the garden minus the TN mass that flowed out of the garden. To test the hypothesis that plants assimilate stormwater N, 4 g of 100 atom% 15N NH4NO3 tracer was used as the N source in the synthetic stormwater during the first 2,000 l synthetic storm event in May. This isotopic label was calculated to enrich the total N pool of each garden to 100‰ 15N/14Nair. New growth was harvested from each plant in both cells and analyzed for 15N before the isotopic label was introduced and weekly thereafter. In May 2012, the upland garden captured 6.2 grams of TN from the added stormwater (55% of TN added), and the wetland garden captured 7.1 grams of TN from the added stormwater (67% of TN added). Within two weeks of adding the label, the 15N ratio increased 500‰ to 3,000‰ in all plant tissues tested in both systems. The results of the isotopic labeling experiment support the hypothesis that the plants used in both vegetated bioretention systems directly contribute to stormwater N treatment through N assimilation.

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

NASA Astrophysics Data System (ADS)

Vugmeyster, Liliya; Ostrovsky, Dmitry; Fu, Riqiang

2015-10-01

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

Identification of microbial populations assimilating nitrogen from RDX in munitions contaminated military training range soils by high sensitivity stable isotope probing.

PubMed

Andeer, Peter; Stahl, David A; Lillis, Lorraine; Strand, Stuart E

2013-09-17

The leaching of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) from particulates deposited in live-fire military training range soils contributes to significant pollution of groundwater. In situ microbial degradation has been proposed as a viable method for onsite containment of RDX. However, there is only a single report of RDX degradation in training range soils and the soil microbial communities involved in RDX degradation were not identified. Here we demonstrate aerobic RDX degradation in soils taken from a target area of an Eglin Air Force Base bombing range, C52N Cat's Eye, (Eglin, Florida U.S.A.). RDX-degradation activity was spatially heterogeneous (found in less than 30% of initial target area field samples) and dependent upon the addition of exogenous carbon sources to the soils. Therefore, biostimulation (with exogenous carbon sources) and bioaugmentation may be necessary to sustain timely and effective in situ microbial biodegradation of RDX. High sensitivity stable isotope probing analysis of extracted soils incubated with fully labeled (15)N-RDX revealed several organisms with (15)N-labeled DNA during RDX-degradation, including xplA-bearing organisms. Rhodococcus was the most prominent genus in the RDX-degrading soil slurries and was completely labeled with (15)N-nitrogen from the RDX. Rhodococcus and Williamsia species isolated from these soils were capable of using RDX as a sole nitrogen source and possessed the genes xplB and xplA associated with RDX-degradation, indicating these genes may be suitable genetic biomarkers for assessing RDX degradation potential in soils. Other highly labeled species were primarily Proteobacteria, including: Mesorhizobium sp., Variovorax sp., and Rhizobium sp.

Comparing five front-of-pack nutrition labels' influence on consumers' perceptions and purchase intentions.

PubMed

Gorski Findling, Mary T; Werth, Paul M; Musicus, Aviva A; Bragg, Marie A; Graham, Dan J; Elbel, Brian; Roberto, Christina A

2018-01-01

In 2011, a National Academy of Medicine report recommended that packaged food in the U.S. display a uniform front-of-package nutrition label, using a system such as a 0-3 star ranking. Few studies have directly compared this to other labels to determine which best informs consumers and encourages healthier purchases. In 2013, we randomized adult participants (N=1247) in an Internet-based survey to one of six conditions: no label control; single traffic light; multiple traffic light; Facts Up Front; NuVal; or 0-3 star ranking. We compared groups on purchase intentions and accuracy of participants' interpretation of food labels. There were no differences in the nutritional quality of hypothetical shopping baskets across conditions (p=0.845). All labels improved consumers' abilities to judge the nutritional quality of foods relative to no label, but the best designs varied by outcomes. NuVal and multiple traffic light labels led to the greatest accuracy identifying the healthier of two products (p<0.001), while the multiple traffic light also led to the most accurate estimates of saturated fat, sugar, and sodium (p<0.001). The single traffic light outperformed other labels when participants compared nutrient levels between similar products (p<0.03). Single/multiple traffic light and Facts Up Front labels led to the most accurate calories per serving estimations (p<0.001). Although front-of-package labels helped participants more accurately assess products' nutrition information relative to no label, no conditions shifted adults' purchase intentions. Results did not point to a clearly superior label design, but they suggest that a 3-star label might not be best for educating consumers. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adelstein, S.J.; Kassis, A.I.

This report describes many factors that need to be considered if radioimmunotherapy is to become a commonplace reality. For beta-emitting radionuclides, two physical features of importance are half-life and energy, with the latter determining the range. These features must be matched to the pharmacokinetics of the carrier and the distribution of the radionuclide, both macroscopically and microscopically. Alpha-particle emitters could be considered for cells that are readily accessible to the labeled antibody and for populations that uniformly and constantly display the targeted antigen or idiotype, e.g., trafficking cells such as T or B lymphocytes. For cells that concentrate the radioactivemore » label, the use of low-energy electrons should be examined. If the radionuclide is translocated to the nucleus, the Auger effect can be particularly lethal because of the high LET-like biological response. 15 refs., 3 figs., 2 tabs.« less

Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra

NASA Astrophysics Data System (ADS)

Gopinath, T.; Veglia, Gianluigi

2013-05-01

We propose a general method that enables the acquisition of multiple 2D and 3D solid-state NMR spectra for U-13C, 15N-labeled proteins. This method, called MEIOSIS (Multiple ExperIments via Orphan SpIn operatorS), makes it possible to detect four coherence transfer pathways simultaneously, utilizing orphan (i.e., neglected) spin operators of nuclear spin polarization generated during 15N-13C cross polarization (CP). In the MEIOSIS experiments, two phase-encoded free-induction decays are decoded into independent nuclear polarization pathways using Hadamard transformations. As a proof of principle, we show the acquisition of multiple 2D and 3D spectra of U-13C, 15N-labeled microcrystalline ubiquitin. Hadamard decoding of CP coherences into multiple independent spin operators is a new concept in solid-state NMR and is extendable to many other multidimensional experiments. The MEIOSIS method will increase the throughput of solid-state NMR techniques for microcrystalline proteins, membrane proteins, and protein fibrils.

Nitrogen transformations following tropical forest felling and burning on a volcanic soil

NASA Technical Reports Server (NTRS)

Matson, Pamela A.; Vitousek, Peter M.; Ewel, John J.; Mazzarino, Maria Julia; Robertson, G. Philip

1987-01-01

Nitrogen transformations and loss were measured following forest clearing in a relatively fertile tropical forest site. Nitrogen mineralization, nitrification, and amounts of ammonium and nitrate increased substantially in surface soils during the 6 mo following burning, then returned to background levels. The nitrogen content of microbial biomass declined to half its original value 6 mo after clearing and remained low in the cleared sites. Plant uptake of nitrogen was substantial on cleared plots (50 g/sq m), but it accounted for only 18 percent of N-15 label added to field plots. MIcrobial immobilization of N-15 was small relative to that in a cleared temperate site, and measurements of denitrification potentials suggested that relatively little mineralized nitrogen was lost to the atmosphere. Substantial amounts of nitrogen (40-70 g/sq m) were retained as exchangeably bound nitrate deep in the soils of a cleared plot on which revegetation was prevented; this process accounted for 12 percent of the N-15 label added to field plots.

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution.

PubMed

António, Carla; Päpke, Carola; Rocha, Marcio; Diab, Houssein; Limami, Anis M; Obata, Toshihiro; Fernie, Alisdair R; van Dongen, Joost T

2016-01-01

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [(13)C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [(13)C]glutamate and [(15)N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the (13)C/(15)N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia. © 2016 American Society of Plant Biologists. All Rights Reserved.

Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.

PubMed

Basu, Sankha S; Mesaros, Clementina; Gelhaus, Stacy L; Blair, Ian A

2011-02-15

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

Water-Solubilized, Cap-Stabilized, Helical Polyalanines: Calibration Standards for NMR and CD Analyses

PubMed Central

Heitmann, Björn; Job, Gabriel E.; Kennedy, Robert J.; Walker, Sharon M.; Kemp, Daniel S.

2006-01-01

NMR and CD studies are reported for two length series of solubilized, spaced, highly helical polyalanines that are N-capped by the optimal helix stabilizer βAsp-Hel and C-capped by β-aminoalanine beta and that are studied in water at 2 °C, pH 1–8. NMR analysis yields a structural characterization of the peptide AcβAspHelAla8betaNH2 and selected members of one βAspHelAlanbeta series. At pH > 4.5 the βAspHel cap provides a preorganized triad of carboxylate anion and two amide residues that is complementary to the helical polyalanine N-terminus. The C-terminal β-aminoalanine assumes a helix-stabilizing conformation consistent with literature precedents. H(N)CO NMR experiments applied to capped, uniformly 13C- and 15N-labeled Ala8 and Ala12 peptides define Alan hydrogen bonding signatures as α-helical without detectable 310 character. Relative NH→ND exchange rates yield site protection factors PFi that define uniquely high fractional helicities FH for the peptide Alan regions. These Alan calibration series, studied in water and lacking helix-stabilizing tertiary structure, yield the first 13C NMR chemical shifts, 3JHNHα coupling constants, and CD ellipticities [θMolar]λ,n characteristic of a fully helical alanine within an Alan context. CD data are used to assign parameters X and [θ]λ,∞, required for rigorous calculation of FH values from CD ellipticities. PMID:15701003

Application of stable‐isotope labelling techniques for the detection of active diazotrophs

PubMed Central

Angel, Roey; Panhölzl, Christopher; Gabriel, Raphael; Herbold, Craig; Wanek, Wolfgang; Richter, Andreas; Eichorst, Stephanie A.

2017-01-01

Summary Investigating active participants in the fixation of dinitrogen gas is vital as N is often a limiting factor for primary production. Biological nitrogen fixation is performed by a diverse guild of bacteria and archaea (diazotrophs), which can be free‐living or symbionts. Free‐living diazotrophs are widely distributed in the environment, yet our knowledge about their identity and ecophysiology is still limited. A major challenge in investigating this guild is inferring activity from genetic data as this process is highly regulated. To address this challenge, we evaluated and improved several 15N‐based methods for detecting N2 fixation activity (with a focus on soil samples) and studying active diazotrophs. We compared the acetylene reduction assay and the 15N2 tracer method and demonstrated that the latter is more sensitive in samples with low activity. Additionally, tracing 15N into microbial RNA provides much higher sensitivity compared to bulk soil analysis. Active soil diazotrophs were identified with a 15N‐RNA‐SIP approach optimized for environmental samples and benchmarked to 15N‐DNA‐SIP. Lastly, we investigated the feasibility of using SIP‐Raman microspectroscopy for detecting 15N‐labelled cells. Taken together, these tools allow identifying and investigating active free‐living diazotrophs in a highly sensitive manner in diverse environments, from bulk to the single‐cell level. PMID:29027346

77 FR 76824 - Uniform Compliance Date for Food Labeling Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-31

... (FSIS) is establishing January 1, 2016, as the uniform compliance date for new meat and poultry product... This final rule establishes January 1, 2016, as the uniform compliance date for new meat and poultry... consumers in the form of higher prices. FSIS encourages meat and poultry companies to comply with new...

Recoil-Implantation Of Multiple Radioisotopes Towards Wear Rate Measurements And Particle Tracing In Prosthetic Joints

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Jacob A.; Timmers, Heiko; Smith, Paul N.

2011-06-01

This study demonstrates a new method of radioisotope labeling of ultra-high molecular weight polyethylene inserts in prosthetic joints for wear studies. The radioisotopes {sup 97}Ru, {sup 100}Pd, {sup 100}Rh, and {sup 101m}Rh are produced in fusion evaporation reactions induced by {sup 12}C ions in a {sup 92}Zr target foil. The fusion products recoil-implant into ultra-high molecular weight polyethylene plugs, machined to fit into the surface of the inserts. During laboratory simulations of the joint motion, a wear rate of the labeled polyethylene may be measured and the pathways of wear debris particles can be traced by detecting characteristic gamma-rays. Themore » concentration profiles of the radioisotopes extend effectively uniformly from the polyethylene surface to a depth of about 4 {mu}m. The multiplicity of labeling and the use of several gamma-ray lines aids with avoiding systematic measurement uncertainties. Two polyethylene plugs were labeled and one was fitted into the surface of the tibial insert of a knee prosthesis, which had been worn in. Actuation over close to 100,000 cycles with a 900 N axial load and a 24 deg. flexion angle removed (14{+-}1)% of the gamma-ray activity from the plug. Most of this activity dispersed into the serum lubricant identifying this as the important debris pathway. Less than 1% activity was transferred to the femoral component of the prosthesis and the measured activity on the tibial tray was insignificant. Assuming uniform wear across the superior surface of the insert, a wear rate of (12{+-}3) mm{sup 3}/Megacycle was determined. This is consistent with wear rate measurements under similar conditions using other techniques.« less

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

NASA Astrophysics Data System (ADS)

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

2014-05-01

Coral reef ecosystems maintain high primary productivity though the seawater is extremely oligotrophic. One of the hypotheses to explain this paradox is the recycling of nutrients in animal-algal symbiotic organisms such as corals. It is relatively easy to measure nutrient uptake rates by corals from seawater, but the proportion of internally circulating nutrients between the coral host and the endosymbiotic algae (zooxanthellae) is more challenging. Here, we performed a long-term and continuous 15N-labelling experiment to quantify the proportionate contribution of seawater (external N source) and the animal host (internal N source) to the total N influx in the endosymbiotic algae. Branches from the scleractinian corals Porites cylindrica and Montipora digitata from Okinawa, Japan, were cultured for 2 months in indoor, flow-through, filtered seawater tanks with the continuous supply of 15N-labelled nitrate. At the initial and after 2, 4, and 9 weeks of the study, coral branches were collected and the algal and animal fractions were separated for isotopic analyses. In both corals, the N isotope ratio of symbiotic algae exponentially increased and the values were much higher than those of the host tissue, suggesting that the algae had a faster turnover N time than the animal host. Algal and host N biomass normalized to the coral surface area slowly decreased in both coral species over the study period. To calculate the contribution of internal and external N, a simple mixing model of algal N metabolism was designed. Using differential equations of 15N balance and N biomass balance, F1 and F2 (external and internal N fluxes to symbiotic algae, respectively) were expressed as the functions of time. The model calculations showed that F2 was much higher than F1 in P. cylindrica and the percentage of internal N to the total influx N (PIN) was >70%. On the other hand, the contribution of F1 and F2 was comparable in M. digitata and the PIN was 40-70%. These results quantitatively showed that the internal N pool in the coral tissue plays an important role in the symbiotic algal metabolism. The application of the present 15N-tracer technique would enable us to further calculate the fluxes of internal and external N in not only corals but also other algal-animal symbiotic organisms under various environmental conditions.

Synthesis of stereoarray isotope labeled (SAIL) lysine via the "head-to-tail" conversion of SAIL glutamic acid.

PubMed

Terauchi, Tsutomu; Kamikawai, Tomoe; Vinogradov, Maxim G; Starodubtseva, Eugenia V; Takeda, Mitsuhiro; Kainosho, Masatsune

2011-01-07

A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6-(2)H(4);1,2,3,4,5,6-(13)C(6);2,6-(15)N(2)]lysine, was synthesized by the "head-to-tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4-(2)H(2);1,2,3,4,5-(13)C(5);2-(15)N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins.

Nitrogen transformations in a waterhyacinth-based water treatment system. [Eichornia crassipes (Mart. ) Solms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moorhead, K.K.; Reddy, K.R.; Graetz, D.A.

Fate of added /sup 15/NH/sub 4/-N and /sup 15/NO/sub 3/-N in waterhyacinth (Eichhornia crassipes (Mart.) Solms)-based water treatment system was evaluated under controlled conditions. Labeled /sup 15/NH/sub 4/-N uptake by waterhyacinth exceeded /sup 15/NO/sub 3/-N uptake. Total /sup 15/N recovery by waterhyacinth ranged from 57 to 72% for added /sup 15/NO/sub 3/-N and 70 to 89% for added /sup 15/NH/sub 4/-N. Both sediment and detritus were potential sources of N for waterhyacinths. Waterhyacinths cultured in sewage effluent removed 55% of the added /sup 15/NH/sub 4/-N and 14% of the added /sup 15/NO/sub 3/-N, respectively. Three to 44% of the addedmore » /sup 15/NH/sub 4/-N was lost through nitrification in the water column and subsequent denitrification in the underlying sediments, whereas 24 to 86% of the added /sup 15/NO/sub 3/-N was lost through denitrification. In a system without plants, 13 to 89% of the added /sup 15/NH/sub 4/-N and 48 to 96% of the added /sup 15/NO/sub 3/-N were lost from the system through a combination of nitrification/denitrification and NH/sub 3/ volatilization.« less

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

NASA Astrophysics Data System (ADS)

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

2014-12-01

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

Fate of organo-mineral particles in streams: Microbial degradation by streamwater & biofilm assemblages

NASA Astrophysics Data System (ADS)

Hunter, W. R.; Raich, M.; Wanek, W.; Battin, T. J.

2013-12-01

Inland waters are of global biogeochemical importance. They receive carbon inputs of ~ 4.8 Pg C/ y of which, 12 % is buried, 18 % transported to the oceans, and 70 % supports aquatic secondary production. However, the mechanisms that determine the fate of organic matter (OM) in these systems are poorly defined. One aspect of this is the formation of organo-mineral complexes in aquatic systems and their potential as a route for OM transport and burial vs. their use as carbon (C) and nitrogen (N) sources within aquatic systems. Organo-mineral particles form by sorption of dissolved OM to freshly eroded mineral surfaces and may contribute to ecosystem-scale particulate OM fluxes. We experimentally tested the availability of mineral-sorbed OM as a C & N source for streamwater microbial assemblages and streambed biofilms. Organo-mineral particles were constructed in vitro by sorption of 13C:15N-labelled amino acids to hydrated kaolin particles, and microbial degradation of these particles compared with equivalent doses of 13C:15N-labelled free amino acids. Experiments were conducted in 120 ml mesocosms over 7 days using biofilms and water sampled from the Oberer Seebach stream (Austria). Each incubation experienced a 16:8 light:dark regime, with metabolism monitored via changes in oxygen concentrations between photoperiods. The relative fate of the organo-mineral particles was quantified by tracing the mineralization of the 13C and 15N labels and their incorporation into microbial biomass. Here we present the initial results of 13C-label mineralization, incorporation and retention within dissolved organic carbon pool. The results indicate that 514 (× 219) μmol/ mmol of the 13:15N labeled free amino acids were mineralized over the 7-day incubations. By contrast, 186 (× 97) μmol/ mmol of the mineral-sorbed amino acids were mineralized over a similar period. Thus, organo-mineral complexation reduced amino acid mineralization by ~ 60 %, with no differences observed between the streamwater and biofilm assemblages. Throughout the incubations, biofilms were observed to leach dissolved organic carbon (DOC). However, within the streamwater assemblage the presence of both organo-mineral particles and kaolin particles was associated with significant DOC removal (-1.7 % and -7.5 % respectively). Consequently, the study demonstrates that mineral and organo-mineral particles can limit the availability of DOC in aquatic systems, providing nucleation sites for flocculation and fresh mineral surfaces, which facilitate OM-sorption. The formation of these organo-mineral particles subsequently restricts microbial OM degradation, potentially altering the transport and facilitating the burial of OM within streams.

Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments

NASA Astrophysics Data System (ADS)

Sun, Ming-Y. i.; Aller, Robert C.; Lee, Cindy; Wakeham, Stuart G.

2002-06-01

Degradation patterns of sedimentary algal lipids were tracked with time under variable redox treatments designed to mimic conditions in organic-rich, bioturbated deposits. Uniformly 13C-labeled algae were mixed with Long Island Sound surface muddy sediments and exposed to different redox regimes, including continuously oxic and anoxic, and oscillated oxic: anoxic conditions. Concentrations of several 13C-labeled algal fatty acids (16:1, 16:0 and 18:1), phytol and an alkene were measured serially. Results showed a large difference (∼10×) in first-order degradation rate constants of cell-associated lipids between continuously oxic and anoxic conditions. Exposure to oxic conditions increased the degradation of cell-associated lipids, and degradation rate constants were positive functions (linear or nonlinear) of the fraction of time sediments were oxic. Production of two new 13C-labeled compounds (iso-15:0 fatty acid and hexadecanol) further indicated that redox conditions and oxic: anoxic oscillations strongly affect microbial degradation of algal lipids and net synthesis of bacterial biomass. Production of 13C-labeled iso-15:0 fatty acid (a bacterial biomarker) was inversely proportional to the fraction of time sediments were oxic, rapidly decreasing after 10 days of incubation under oxic and frequently oscillated conditions. Turnover of bacterial biomass was faster under continuously or occasionally oxic conditions than under continuously anoxic conditions. 13C-labeled hexadecanol, an intermediate degradation product, accumulated under anoxic conditions but not under oxic or periodically oxic conditions. The frequency of oxic: anoxic oscillation clearly alters both the rate and pathways of lipid degradation in surficial sediments. Terminal degradation efficiency and lipid products from degradation of algal material depend on specific patterns of redox fluctuations.

Flux Analysis of Free Amino Sugars and Amino Acids in Soils by Isotope Tracing with a Novel Liquid Chromatography/High Resolution Mass Spectrometry Platform.

PubMed

Hu, Yuntao; Zheng, Qing; Wanek, Wolfgang

2017-09-05

Soil fluxomics analysis can provide pivotal information for understanding soil biochemical pathways and their regulation, but direct measurement methods are rare. Here, we describe an approach to measure soil extracellular metabolite (amino sugar and amino acid) concentrations and fluxes based on a 15 N isotope pool dilution technique via liquid chromatography and high-resolution mass spectrometry. We produced commercially unavailable 15 N and 13 C labeled amino sugars and amino acids by hydrolyzing peptidoglycan isolated from isotopically labeled bacterial biomass and used them as tracers ( 15 N) and internal standards ( 13 C). High-resolution (Orbitrap Exactive) MS with a resolution of 50 000 allowed us to separate different stable isotope labeled analogues across a large range of metabolites. The utilization of 13 C internal standards greatly improved the accuracy and reliability of absolute quantification. We successfully applied this method to two types of soils and quantified the extracellular gross fluxes of 2 amino sugars, 18 amino acids, and 4 amino acid enantiomers. Compared to the influx and efflux rates of most amino acids, similar ones were found for glucosamine, indicating that this amino sugar is released through peptidoglycan and chitin decomposition and serves as an important nitrogen source for soil microorganisms. d-Alanine and d-glutamic acid derived from peptidoglycan decomposition exhibited similar turnover rates as their l-enantiomers. This novel approach offers new strategies to advance our understanding of the production and transformation pathways of soil organic N metabolites, including the unknown contributions of peptidoglycan and chitin decomposition to soil organic N cycling.

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry.

PubMed

Poe, Bobby G; Navratil, Marian; Arriaga, Edgar A

2006-12-29

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.

Differential ammonia metabolism in Aedes aegypti fat body and midgut tissues

PubMed Central

Scaraffia, Patricia Y.; Zhang, Quigfen; Thorson, Kelsey; Wysocki, Vicki H.; Miesfeld, Roger L.

2010-01-01

In order to understand at the tissue level how Aedes aegypti copes with toxic ammonia concentrations that result from the rapid metabolism of blood meal proteins, we investigated the incorporation of 15N from 15NH4Cl into amino acids using an in vitro tissue culture system. Fat body or midgut tissues from female mosquitoes were incubated in an Aedes saline solution supplemented with glucose and 15NH4Cl for 10–40 minutes. The media was then mixed with deuterium-labeled amino acids, dried and derivatized. The 15N-labeled and unlabeled amino acids in each sample were quantified by mass spectrometry techniques. The results demonstrate that both tissues efficiently incorporate ammonia into amino acids, however, the specific metabolic pathways are distinct. In the fat body, the 15N from 15NH4Cl is first incorporated into the amide side chain of Gln and then into the amino group of Gln, Glu, Ala and Pro. This process mainly occurs via the glutamine synthetase (GS) and glutamate synthase (GltS) pathway. In contrast, 15N in midgut is first incorporated into the amino group of Glu and Ala, and then into the amide side chain of Gln. Interestingly, our data show that the GS/GltS pathway is not functional in the midgut. Instead, midgut cells detoxify ammonia by glutamate dehydrogenase, alanine aminotransferase and GS. These data provide new insights into ammonia metabolism in A. aegypti mosquitoes. PMID:20206632

Engineering soil organic matter quality: Biodiesel Co-Product (BCP) stimulates exudation of nitrogenous microbial biopolymers

PubMed Central

Redmile-Gordon, Marc A.; Evershed, Richard P.; Kuhl, Alison; Armenise, Elena; White, Rodger P.; Hirsch, Penny R.; Goulding, Keith W.T.; Brookes, Philip C.

2015-01-01

Biodiesel Co-Product (BCP) is a complex organic material formed during the transesterification of lipids. We investigated the effect of BCP on the extracellular microbial matrix or ‘extracellular polymeric substance’ (EPS) in soil which is suspected to be a highly influential fraction of soil organic matter (SOM). It was hypothesised that more N would be transferred to EPS in soil given BCP compared to soil given glycerol. An arable soil was amended with BCP produced from either 1) waste vegetable oils or 2) pure oilseed rape oil, and compared with soil amended with 99% pure glycerol; all were provided with 15N labelled KNO3. We compared transfer of microbially assimilated 15N into the extracellular amino acid pool, and measured concomitant production of exopolysaccharide. Following incubation, the 15N enrichment of total hydrolysable amino acids (THAAs) indicated that intracellular anabolic products had incorporated the labelled N primarily as glutamine and glutamate. A greater proportion of the amino acids in EPS were found to contain 15N than those in the THAA pool, indicating that the increase in EPS was comprised of bioproducts synthesised de novo. Moreover, BCP had increased the EPS production efficiency of the soil microbial community (μg EPS per unit ATP) up to approximately double that of glycerol, and caused transfer of 21% more 15N from soil solution into EPS-amino acids. Given the suspected value of EPS in agricultural soils, the use of BCP to stimulate exudation is an interesting tool to consider in the theme of delivering sustainable intensification. PMID:26635420

Estimate of production of gaseous nitrogen in the human body based on (15)N analysis of breath N2 after administration of [(15)N2]urea.

PubMed

Junghans, Peter

2013-01-01

After oral administration of [(15)N2]urea (1.5 mmol, 95 atom% (15)N), we found that breath N2 was significantly (15)N-labelled. The result suggests that molecular nitrogen in breath must be partly produced endogenously. Based on a metabolic model, the endogenous N2 production was estimated to be 0.40±0.25 mmol kg(-1) d(-1) or 2.9±1.8 % of the total (urinary and faecal) N excretion in fasted healthy subjects (n=4). In patients infected with Helicobacter pylori (n=5), the endogenous N2 production was increased to 1.24±0.59 mmol kg(-1) d(-1) or 9.0±4.3 % of the total N excretion compared to the healthy controls (p<0.05). We conclude that N balance and gas exchange measurements may be affected by endogenously produced nitrogen, especially in metabolic situations with elevated nitrosation, for instance in oxidative and nitrosative stress-related diseases such as H. pylori infections.

Factual text and emotional pictures: overcoming a false dichotomy of cigarette warning labels.

PubMed

Popova, Lucy; Owusu, Daniel; Jenson, Desmond; Neilands, Torsten B

2017-04-20

In reviewing the first set of pictorial warning labels in the USA, the courts equated textual labels with facts and information, and images with emotion. This study tested the differences in perceived informativeness and emotion between textual and pictorial cigarette warning labels. An online study with 1838 US adults who were non-smokers (n=764), transitioning smokers (quit smoking in the past 2 years or currently trying to quit, n=505) or current smokers (n=569). Each participant evaluated 9 out of 81 text and pictorial cigarette warning labels. Participants reported to what extent they perceived the label as informative and factual and the negative emotions they felt while looking at each label. We used linear mixed models to account for the nesting of multiple observations within each participant. There were no significant differences in perceived informativeness between textual (mean 6.15 on a 9-point scale) and pictorial labels (6.14, p=0.80, Cohen's d=0.003). Textual labels evoked slightly less emotion (4.21 on a 9-point scale) than pictorial labels (4.42, p<0.001, Cohen's d=0.08). Perceived informativeness and emotion were strongly correlated (Pearson r=0.53, p<0.001). Our findings contradict courts' conclusions that pictorial messages are emotional and not factual. Pictorial labels are rated as informative and factual, textual labels evoke emotion, and emotionality and informativeness are strongly correlated. These findings serve as evidence for the Food and Drug Administration (FDA) to counteract the claim that pictorial warning labels, by definition, are not 'purely factual and uncontroversial'. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The fate of nitrogen mineralized from leaf litter — Initial evidence from 15N-labeled litter

Treesearch

Kathryn B. Piatek

2011-01-01

Decomposition of leaf litter includes microbial immobilization of nitrogen (N), followed by N mineralization. The fate of N mineralized from leaf litter is unknown. I hypothesized that N mineralized from leaf litter will be re-immobilized into other forms of organic matter, including downed wood. This mechanism may retain N in some forests. To test this hypothesis, oak...

16 CFR 305.11 - Labeling for refrigerators, refrigerator-freezers, freezers, dishwashers, clothes washers, water...

Code of Federal Regulations, 2011 CFR

2011-01-01

... headline, copy, and charts to maintain uniformity for immediate consumer recognition and readability. Trim... faces to be used are indicated on the prototype labels. No hyphenation should be used in setting...

16 CFR 305.11 - Labeling for refrigerators, refrigerator-freezers, freezers, dishwashers, clothes washers, water...

Code of Federal Regulations, 2010 CFR

2010-01-01

... headline, copy, and charts to maintain uniformity for immediate consumer recognition and readability. Trim... faces to be used are indicated on the prototype labels. No hyphenation should be used in setting...

16 CFR 305.11 - Labeling for refrigerators, refrigerator-freezers, freezers, dishwashers, clothes washers, water...

Code of Federal Regulations, 2012 CFR

2012-01-01

... headline, copy, and charts to maintain uniformity for immediate consumer recognition and readability. Trim... faces to be used are indicated on the prototype labels. No hyphenation should be used in setting...

16 CFR 305.11 - Labeling for refrigerators, refrigerator-freezers, freezers, dishwashers, clothes washers, water...

Code of Federal Regulations, 2013 CFR

2013-01-01

... headline, copy, and charts to maintain uniformity for immediate consumer recognition and readability. Trim... faces to be used are indicated on the prototype labels. No hyphenation should be used in setting...

Measurement of protein digestibility in humans by a dual-tracer method.

PubMed

Devi, Sarita; Varkey, Aneesia; Sheshshayee, M S; Preston, Thomas; Kurpad, Anura V

2018-06-01

Recent evaluations of the risk of dietary protein deficiency have indicated that protein digestibility may be a key limiting factor in the provision of indispensable amino acids (IAAs), particularly for vulnerable populations living in challenging environments where intestinal dysfunction may exist. Since the digestion of protein occurs only in the small intestine, and the metabolic activity of colonic bacteria confounds measurements at the fecal level, there is a need to develop noninvasive protein digestibility measurements at the ileal level. We used a dual-tracer method with stable isotopes to characterize the digestibility of uniformly labeled [13C]-spirulina protein as a standard protein, in comparison to a mixture of 2H-labeled crystalline amino acids, and then demonstrated the use of this standard protein to measure the digestibility of selected legumes (chick pea and mung bean) through the use of proteins that were intrinsically labeled with 2H. The digestibility of uniformly labeled [13C]-spirulina was first measured in 6 healthy volunteers (3 males and 3 females) by feeding it along with a standard mixture of 2H-labeled amino acids, in a dual-tracer, plateau-fed test meal approach. Next, intrinsically labeled legume protein digestibility was studied with a similar dual-tracer approach, with uniformly labeled [13C]-spirulina as the standard, when processed differently before consumption. The average digestibility of IAA in spirulina protein was 85.2%. The average IAA digestibility of intrinsically 2H-labeled chick pea and mung bean protein was 56.6% and 57.7%, respectively. Dehulling of mung bean before ingestion increased the average IAA digestibility by 9.9% in comparison to whole mung bean digestibility. An innovative, minimally invasive "dual-stable-isotope" method was developed to measure protein digestibility, in which the ingestion of an intrinsically 2H-labeled test protein along with a 13C-labeled standard protein of known digestibility allows for an accurate measure of digestion and absorption of the intrinsically labeled protein. This minimally invasive method is critical to redefining protein quality and will aid in revisiting human protein requirements in different settings and in vulnerable populations. This trial was registered at Clinical Trials Registry-India as CTRI/2017/11/010468.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

PubMed

Mavri-Damelin, Demetra; Damelin, Leonard H; Eaton, Simon; Rees, Myrddin; Selden, Clare; Hodgson, Humphrey J F

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Relative quantitation of glycosylation variants by stable isotope labeling of enzymatically released N-glycans using [12C]/[13C] aniline and ZIC-HILIC-ESI-TOF-MS.

PubMed

Giménez, Estela; Sanz-Nebot, Victòria; Rizzi, Andreas

2013-09-01

Glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline was used for relative quantitation of N-glycans. In a first step, the labeling method by reductive amination was optimized for this reagent. It could be demonstrated that selecting aniline as limiting reactant and using the reductant in excess is critical for achieving high derivatization yields (over 95 %) and good reproducibility (relative standard deviations ∼1-5 % for major and ∼5-10 % for minor N-glycans). In a second step, zwitterionic-hydrophilic interaction liquid chromatography in capillary columns coupled to electrospray mass spectrometry with time-of-flight analyzer (μZIC-HILIC-ESI-TOF-MS) was applied for the analysis of labeled N-glycans released from intact glycoproteins. Ovalbumin, bovine α1-acid-glycoprotein and bovine fetuin were used as test glycoproteins to establish and evaluate the methodology. Excellent separation of isomeric N-glycans and reproducible quantitation via the extracted ion chromatograms indicate a great potential of the proposed methodology for glycoproteomic analysis and for reliable relative quantitation of glycosylation variants in biological samples.

"Passive victim – strong survivor"? Perceived meaning of labels applied to women who were raped

PubMed Central

Papendick, Michael

2017-01-01

Three experiments (total N = 464) were conducted in parallel with English- and German-speaking participants to examine the perceived meanings and effects of the labels "victim" versus "survivor" (and their German equivalents) when applied to a woman who was raped. In Study 1 (N = 179), participants read a rape vignette and then rated the meaning of the label it contained (either "victim" or "survivor") on a 15-item semantic differential. Independent of language and participant gender, "survivor" was perceived more positively overall (e.g., as strong, brave, active) than was "victim" (weak, passive, but also innocent). In Study 2 (N = 95), labels were varied within items assessing judgments of an acquaintance-rape case (e.g., "Does the victim [survivor] … carry a certain responsibility for what happened?"), focusing on short-term outcomes. Significant interaction effects of label and participants’ gender emerged on case-related judgments. Participants in both language samples judged "survivor" to be a less appropriate term than "victim". In Study 3 (N = 190), participants read a text in which a woman who had been raped labeled herself as either "victim" or "survivor", focusing on the coping with sexual violence. As in Study 2, German-language participants showed no significant effects of the label on their case judgments but rejected the term "survivor" as inappropriate; English-language participants, by contrast, perceived the woman describing herself as "survivor" to be more psychologically stable and regarded the use of both labels as appropriate. Results are discussed in terms of their applied relevance for communicating about sexual violence. PMID:28493976

Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2004-06-01

Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

PubMed

Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom

2016-10-10

Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.

Organo-mineral complexation alters carbon and nitrogen cycling in stream microbial assemblages

NASA Astrophysics Data System (ADS)

Hunter, William Ross; Wanek, Wolfgang; Prommer, Judith; Mooshammer, Maria; Battin, Tom

2014-05-01

Inland waters are of global biogeochemical importance receiving carbon inputs of ~ 4.8 Pg C y-1. Of this 12 % is buried, 18 % transported to the oceans, and 70 % supports aquatic secondary production. However, the mechanisms that determine the fate of organic matter (OM) in these systems are poorly defined. One important aspect is the formation of organo-mineral complexes in aquatic systems and their potential as a route for OM transport and burial vs. microbial utilization as organic carbon (C) and nitrogen (N) sources. Organo-mineral particles form by sorption of dissolved OM to freshly eroded mineral surfaces and may contribute to ecosystem-scale particulate OM fluxes. We tested the availability of mineral-sorbed OM as a C & N source for streamwater microbial assemblages and streambed biofilms. Organo-mineral particles were constructed in vitro by sorption of 13C:15N-labelled amino acids to hydrated kaolin particles, and microbial degradation of these particles compared with equivalent doses of 13C:15N-labelled free amino acids. Experiments were conducted in 120 ml mesocosms over 7 days using biofilms and streamwater sampled from the Oberer Seebach stream (Austria), tracing assimilation and mineralization of 13C and 15N labels from mineral-sorbed and dissolved amino acids. Here we present data on the effects of organo-mineral sorption upon amino acid mineralization and its C:N stoichiometry. Organo-mineral sorption had a significant effect upon microbial activity, restricting C and N mineralization by both the biofilm and streamwater treatments. Distinct differences in community response were observed, with both dissolved and mineral-stabilized amino acids playing an enhanced role in the metabolism of the streamwater microbial community. Mineral-sorption of amino acids differentially affected C & N mineralization and reduced the C:N ratio of the dissolved amino acid pool. The present study demonstrates that organo-mineral complexes restrict microbial degradation of OM and may, consequently, alter the carbon and nitrogen cycling dynamics within aquatic ecosystems.

Molecular understanding of Abeta peptide interaction with isoflurane, propofol, and thiopental: NMR spectroscopic study.

PubMed

Mandal, Pravat K; Williams, John P; Mandal, Ratna

2007-01-23

Abeta peptide is the major component of senile plaques (SP), which accumulate in the brain of a patient with Alzheimer's disease (AD). A recent report indicated that isoflurane enhanced Abeta oligomerization (micro-aggregation) and subsequent cytotoxicity of the Abeta peptide. A separate study showed that a clinically relevant concentration of isoflurane induces apoptosis and increases Abeta production in a human neuroglioma cell line. In vitro studies have indicated that halothane interacts specifically with Abeta peptide to induce oligomerization and that Abeta42 oligomerizes faster than Abeta40. The specific interactions of isoflurane, propofol, and thiopental with uniformly 15N labeled Abeta40 and Abeta42 peptide were investigated using multidimensional nuclear magnetic resonance (NMR) experiments. We found that isoflurane and propofol (at higher concentration) interact with Abeta40 peptides and induce Abeta oligomerization. Thiopental does not interact with specific residues (G29, A30, and I31) of Abeta40; hence, the peptide remains in the monomeric form. On the basis of our NMR study, thiopental does not oligomerize Abeta40 even at higher concentrations.

Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

PubMed Central

Drummond, Meghan C.; Barzik, Melanie; Bird, Jonathan E.; Zhang, Duan-Sun; Lechene, Claude P.; Corey, David P.; Cunningham, Lisa L.; Friedman, Thomas B.

2015-01-01

The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips. PMID:25898120

Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex.

PubMed

Jiang, F; Kumar, R A; Jones, R A; Patel, D J

1996-07-11

The catalytic properties of RNA and its well known role in gene expression and regulation are the consequence of its unique solution structures. Identification of the structural determinants of ligand recognition by RNA molecules is of fundamental importance for understanding the biological functions of RNA, as well as for the rational design of RNA Sequences with specific catalytic activities. Towards this latter end, Szostak et al. used in vitro selection techniques to isolate RNA sequences ('aptamers') containing a high-affinity binding site for ATP, the universal currency of cellular energy, and then used this motif to engineer ribozymes with polynucleotide kinase activity. Here we present the solution structure, as determined by multidimensional NMR spectroscopy and molecular dynamics calculations, of both uniformly and specifically 13C-, 15N-labelled 40-mer RNA containing the ATP-binding motif complexed with AMP. The aptamer adopts an L-shaped structure with two nearly orthogonal stems, each capped proximally by a G x G mismatch pair, binding the AMP ligand at their junction in a GNRA-like motif.

Simulation with models of increasing complexity of CO2 emissions and nitrogen mineralisation, after soil application of labelled pig slurry and maize stalks

NASA Astrophysics Data System (ADS)

Bechini, Luca; Marino Gallina, Pietro; Geromel, Gabriele; Corti, Martina; Cavalli, Daniele

2015-04-01

High amounts of nitrogen are available per unit area in regions with intensive livestock operations. In swine farms, pig slurries are frequently incorporated in the soil together with maize stalks. Simulation models may help to understand nitrogen dynamics associated with animal manure and crop residue decomposition in the soil, and to support the definition of best management practices. The objective of this work was to test the ability of different models to simulate CO2 emissions and nitrogen mineralisation during a laboratory incubation (under optimal soil water content and constant temperature) of maize stalks (ST) and pig slurry (PS). A loam soil was amended with labelled (15N) or unlabelled maize stalks and pig slurries, in the presence of ammonium sulphate (AS). These treatments were established: unfertilised soil; ST15 + AS + PS; ST + AS15 + PS; and ST + AS + PS15. During 180 days, we measured CO2 emissions; microbial biomass C, N, and 15N; and soil mineral N (SMN and SM-15N). Three models of increasing complexity were calibrated using measured data. The models were two modifications of ICBM 2B/N (Kätterer and Andrén, 2001) and CN-SIM (Petersen et al., 2005). The three models simulated rather accurately the emissions of CO2 throughout the incubation period (Relative Root Mean Squared Error, RRMSE = 8-25). The simplest model (with one pool for ST and one for PS) strongly overestimated SMN immobilisation from day 3 to day 21, both in the treatments with AS15 and PS15 (RRMSE = 27-30%). The other two models represented rather well the dynamics of SMN in the soil (RRMSE = 21-25%), simulating a fast increase of nitrate concentration in the first days, and slower rates of nitrification thereafter. Worse performances were obtained with all models for the simulation of SM-15N in the treatment with ST15 (RRMSE = 64-104%): experimental data showed positive mineralization of stalk-derived N from the beginning of the incubation, while models strongly underestimated ST15 mineralisation until day 7. Due to model structure, trade-offs exist between a good simulation of CO2 emissions and a good simulation of SMN. Therefore, simulation performances of the three models are a compromise between the errors in the simulation of C and N dynamics. Thus, some models (especially the simplest one), overestimated or underestimated SMN to match CO2 measurements. This preliminary work emphasised the importance of testing models with both C and N measurements. This reduced the risk of obtaining model parameters suitable for the simulation of N (or opposite C) dynamics that lead to unrealistic simulation of C (or N) decomposition. The use of 15N-labelled materials will help to improve models for the simulation of added organic matter decomposition. Kätterer, T., Andrén, O., 2001. The ICBM family of analytically solved models of soil carbon, nitrogen and microbial biomass dynamics'descriptions and application examples. Ecol. Model. 136, 191-207. doi:10.1016/S0304-3800(00)00420-8. Petersen, B.M., Jensen, L.S., Hansen, S., Pedersen, A., Henriksen, T.M., Sørensen, P., Trinsoutrot-Gattin, I., Berntsen, J., 2005. CN-SIM: a model for the turnover of soil organic matter. II. Short-term carbon and nitrogen development. Soil Biol. Biochem. 37, 375-393. doi:10.1016/j.soilbio.2004.08.007.

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

PubMed

Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

1984-07-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.

PubMed

Ono, Katsuhiko; Jung, Minkyung; Zhang, Tianli; Tsutsuki, Hiroyasu; Sezaki, Hiroshi; Ihara, Hideshi; Wei, Fan-Yan; Tomizawa, Kazuhito; Akaike, Takaaki; Sawa, Tomohiro

2017-05-01

Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34 S-labeled L-cysteine from O-acetyl-L-serine and 34 S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur ( 34 S) and nitrogen ( 15 N) atoms was also achieved by performing enzyme reactions with 15 N-labeled L-serine, acetyl-CoA, and 34 S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34 S-labeled N-acetyl-L-cysteine (NAC) by incubating 34 S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34 S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology. Copyright © 2017 Elsevier Inc. All rights reserved.

S-Methylmethionine Conversion to Dimethylsulfoniopropionate: Evidence for an Unusual Transamination Reaction.

PubMed Central

Rhodes, D.; Gage, D. A.; Cooper, AJL.; Hanson, A. D.

1997-01-01

Leaves of Wollastonia biflora (L.) DC. synthesize the osmoprotectant 3-dimethylsulfoniopropionate (DMSP) from methionine via S-methylmethionine (SMM) and 3-dimethylsulfoniopropionaldehyde (DMSP-ald); no other intermediates have been detected. To test whether the amino group of SMM is lost by transamination or deamination, [methyl-2H3,15N]SMM was supplied to leaf discs, and 15N-labeling of amino acids was monitored, along with synthesis of [2H3]DMSP. After short incubations more 15N was incorporated into glutamate than into other amino acids, and the 15N abundance in glutamate exceeded that in the amide group of glutamine (Gln). This is more consistent with transamination than deamination, because deamination would be predicted to give greater labeling of Gln amide N due to reassimilation, via Gln synthetase, of the 15NH4+ released. This prediction was borne out by control experiments with 15NH4Cl. The transamination product of SMM, 4-dimethylsulfonio-2-oxobutyrate (DMSOB), is expected to be extremely unstable. This was confirmed by attempting to synthesize it enzymatically from SMM using L-amino acid oxidase or Gln transaminase K and from 4-methylthio-2-oxobutyrate using methionine S-methyltransferase. In each case, the reaction product decomposed rapidly, releasing dimethylsulfide. The conversion of SMM to DMSP-ald is therefore unlikely to involve a simple transamination that generates free DMSOB. Plausible alternatives are that DMSOB is channeled within a specialized transaminase-decarboxylase complex or that it exists only as the bound intermediate of a single enzyme catalyzing an unusual transamination-decarboxylation reaction. PMID:12223879

The likelihood of sunburn in sunscreen users is disproportionate to the SPF.

PubMed

Pissavini, Marc; Diffey, Brian

2013-06-01

Sunburn is a common feature in sunscreen users. The purpose of this paper is to estimate the expected frequency and magnitude of sunburn resulting from typical use of sunscreens labelled SPF15 and SPF30 by people spending long periods outdoors in strong summer sunshine. By combining the probability distribution of the measured sun protection factor (SPF) in vivo with those for the average application thickness and the uniformity of application over the skin surface, a simulation model was developed to estimate the variation in delivered protection over the exposed skin surface from consumer use of sunscreens. While either sunscreen, if delivering the nominal SPF over the entire exposed skin, would be sufficient to prevent any erythema, the simulation indicates that the combination of the average quantity applied with the variability in thickness over the skin surface will lead to erythema, especially in SPF15 sunscreen users. People who intend spending long periods outside in strong sunshine would be better advised to use SPF30 labelled sunscreens than SPF15 sunscreens, and to apply the product carefully over exposed skin if they wish to minimize their risk of sunburn and, by implication, skin cancer. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Making Polymeric Microspheres

NASA Technical Reports Server (NTRS)

Rhim, Won-Kyu; Hyson, Michael T.; Chung, Sang-Kun; Colvin, Michael S.; Chang, Manchium

1989-01-01

Combination of advanced techniques yields uniform particles for biomedical applications. Process combines ink-jet and irradiation/freeze-polymerization techniques to make polymeric microspheres of uniform size in diameters from 100 to 400 micrometer. Microspheres used in chromatography, cell sorting, cell labeling, and manufacture of pharmaceutical materials.

Hydrogen Exchange During Cell-Free Incorporation of Deuterated Amino Acids and an Approach to its Inhibition

PubMed Central

Tonelli, M.; Singarapu, K. K.; Markley, J. L.; Makino, S.; Sahu, S .C.; Matsubara, Y.; Endo, Y.; Kainosho, M.

2012-01-01

Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H2O, exchange reactions can lead to contamination of 2H sites by 1H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing 1H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM L-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-2H,15N]-chlorella ubiquitin without and with added inhibitors, and [U-15N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-13C,15N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at Cα sites with the exception of Gly. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Ala-Hβ, Asn-Hβ, Asp-Hβ, Gln-Hγ, Glu-Hγ, and Lys-Hε. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of interest in studies of large proteins, protein complexes, and membrane proteins. PMID:21984356

Synthesis and Labeling of RNA In Vitro

PubMed Central

Huang, Chao; Yu, Yi-Tao

2013-01-01

This unit discusses several methods for generating large amounts of uniformly labeled, end-labeled, and site-specifically labeled RNAs in vitro. The methods involve a number of experimental procedures, including RNA transcription, 5′ dephosphorylation and rephosphorylation, 3′ terminal nucleotide addition (via ligation), site-specific RNase H cleavage directed by 2′-O-methyl RNA-DNA chimeras, and 2-piece splint ligation. The applications of these RNA radiolabeling approaches are also discussed. PMID:23547015

A traffic light food labeling intervention increases consumer awareness of health and healthy choices at the point-of-purchase.

PubMed

Sonnenberg, Lillian; Gelsomin, Emily; Levy, Douglas E; Riis, Jason; Barraclough, Susan; Thorndike, Anne N

2013-10-01

We surveyed customers in a hospital cafeteria in Boston, Massachusetts before and after implementation of traffic light food labeling to determine the effect of labels on customers' awareness and purchase of healthy foods. Cafeteria items were identified as red (unhealthy), yellow (less healthy), or green (healthy). Customers were interviewed before (N=166) and after (N=223) labeling was implemented. Each respondent was linked to cash register data to determine the proportion of red, yellow, and green items purchased. Data were collected from February-April 2010. We compared responses to survey questions and mean proportion of red, yellow, and green items per transaction between customers interviewed during baseline and customers interviewed during the intervention. Survey response rate was 60%. Comparing responses during labeling intervention to baseline, more respondents identified health/nutrition as an important factor in their purchase (61% vs. 46%, p=0.004) and reported looking at nutrition information (33% vs. 15%, p<0.001). Respondents who noticed labels during the intervention and reported that labels influenced their purchases were more likely to purchase healthier items than respondents who did not notice labels (p<0.001 for both). Traffic light food labels prompted individuals to consider their health and to make healthier choices at point-of-purchase. © 2013.

"Afterlife experiment": use of MALDI-MS and SIMS imaging for the study of the nitrogen cycle within plants.

PubMed

Seaman, Callie; Flinders, Bryn; Eijkel, Gert; Heeren, Ron M A; Bricklebank, Neil; Clench, Malcolm R

2014-10-21

As part of a project to demonstrate the science of decay, a series of mass spectrometry imaging experiments were performed. The aim was to demonstrate that decay and decomposition are only part of the story and to show pictorially that atoms and molecules from dead plants and animals are incorporated into new life. Radish plants (Raphanus sativus) were grown hydroponically using a nutrient system containing (15)N KNO3 (98% labeled) as the only source of nitrogen. Plants were cropped and left to ferment in water for 2 weeks to create a radish "tea", which was used as a source of nitrogen for radish grown in a second hydroponics experiment. After 5 weeks of growth, the radish plants were harvested and cryosectioned, and sections were imaged by positive-ion MALDI and SIMS mass spectrometry imaging. The presence of labeled species in the plants grown using (15)N KNO3 as nutrient and those grown from the radish "tea" was readily discernible. The uptake of (15)N into a number of identifiable metabolites has been studied by MALDI-MS and SIMS imaging.

Formation of carbonato and hydroxo complexes in the reaction of platinum anticancer drugs with carbonate.

PubMed

Di Pasqua, Anthony J; Centerwall, Corey R; Kerwood, Deborah J; Dabrowiak, James C

2009-02-02

The second-generation Pt(II) anticancer drug carboplatin is here shown to react with carbonate, which is present in blood, interstitial fluid, cytosol, and culture medium, to produce platinum-carbonato and -hydroxo complexes. Using [(1)H-(15)N] HSQC NMR and (15)N-labeled carboplatin, we observe that cis-[Pt(CBDCA-O)(OH)(NH(3))(2)](-), cis-[Pt(OH)(2)(NH(3))(2)], cis-[Pt(CO(3))(OH)(NH(3))(2)](-), and what may be cis-[Pt(CO(3))(NH(3))(2)] are produced when 1 is allowed to react in 23.8 mM carbonate buffer. When (15)N-labeled carboplatin is allowed to react in 0.5 M carbonate buffer, these platinum species, as well as other hydroxo and carbonato species, some of which may be dinuclear complexes, are produced. Furthermore, we show that the carbonato species cis-[Pt(CO(3))(OH)(NH(3))(2)](-) is also produced when cisplatin is allowed to react in carbonate buffer. The study outlines the conditions under which carboplatin and cisplatin form carbonato and aqua/hydroxo species in carbonate media.

Fluorine-18 labeling of an anti-HER2 VHH using a residualizing prosthetic group via a strain-promoted click reaction: Chemistry and preliminary evaluation.

PubMed

Zhou, Zhengyuan; Chitneni, Satish K; Devoogdt, Nick; Zalutsky, Michael R; Vaidyanathan, Ganesan

2018-05-01

In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with 18 F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-containing residualizing moiety, and an 18 F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb 2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and guanidine functionalities. The thus derivatized sdAbs were radiolabeled with 18 F using an 18 F-labeled aza-dibenzocyclooctyne derivative ([ 18 F]F-ADIBO) via SPAAC, generating the desired conjugate ([ 18 F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [ 18 F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [ 18 F]RL-II-2Rs15d and [ 125 I]SGMIB-2Rs15d, and microPET/CT imaging of [ 18 F]RL-II-2Rs15d and the [ 18 F]RL-II-R3B23 control sdAb were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of 23.9 ± 6.9% (n = 8) was achieved for the SPAAC reaction between [ 18 F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (K d ) and IRF for the binding of [ 18 F]RL-II-2Rs15d to HER2 were 5.6 ± 1.3 nM and 73.1 ± 22.5% (n = 3), respectively. The specific uptake of [ 18 F]RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14-17% of the input dose at 1, 2, and 4 h, slightly higher than seen for co-incubated [ 125 I]SGMIB-2Rs15d. The uptake of [ 18 F]RL-II-2Rs15d in SKOV-3 xenografts at 1 h and 2 h p.i. were 5.54 ± 0.77% ID/g and 6.42 ± 1.70% ID/g, respectively, slightly higher than those for co-administered [ 125 I]SGMIB-2Rs15d (4.80 ± 0.78% ID/g and 4.78 ± 1.39% ID/g). MicroPET/CT imaging with [ 18 F]RL-II-2Rs15d at 1-3 h p.i. clearly delineated SKOV-3 tumors while no significant accumulation of activity in tumor was seen for [ 18 F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [ 18 F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is the first time SPAAC method has been used to label an sdAb with 18 F, especially with residualizing functionality. Copyright © 2018 Elsevier Ltd. All rights reserved.

Allocation of Nitrogen and Carbon Is Regulated by Nodulation and Mycorrhizal Networks in Soybean/Maize Intercropping System

PubMed Central

Wang, Guihua; Sheng, Lichao; Zhao, Dan; Sheng, Jiandong; Wang, Xiurong; Liao, Hong

2016-01-01

Soybean/maize intercropping has remarkable advantages in increasing crop yield and nitrogen (N) efficiency. However, little is known about the contributions of rhizobia or arbuscular mycorrhizal fungi (AMF) to yield increases and N acquisition in the intercropping system. Plus, the mechanisms controlling carbon (C) and N allocation in intercropping systems remain unsettled. In the present study, a greenhouse experiment combined with 15N and 13C labeling was conducted using various inoculation and nutrient treatments. The results showed that co-inoculation with AMF and rhizobia dramatically increased biomass and N content of soybean and maize, and moderate application of N and phosphorus largely amplified the effect of co-inoculation. Maize had a competitive advantage over soybean only under co-inoculation and moderate nutrient availability conditions, indicating that the effects of AMF and rhizobia in intercropping systems are closely related to nutrient status. Results from 15N labeling showed that the amount of N transferred from soybean to maize in co-inoculations was 54% higher than that with AMF inoculation alone, with this increased N transfer partly resulting from symbiotic N fixation. The results from 13C labeling showed that 13C content increased in maize shoots and decreased in soybean roots with AMF inoculation compared to uninoculated controls. Yet, with co-inoculation, 13C content increased in soybean. These results indicate that photosynthate assimilation is stimulated by AM symbiosis in maize and rhizobial symbiosis in soybean, but AMF inoculation leads to soybean investing more carbon than maize into common mycorrhizal networks (CMNs). Overall, the results herein demonstrate that the growth advantage of maize when intercropped with soybean is due to acquisition of N by maize via CMNs while this crop contributes less C into CMNs than soybean under co-inoculation conditions. PMID:28018420

Allocation of Nitrogen and Carbon Is Regulated by Nodulation and Mycorrhizal Networks in Soybean/Maize Intercropping System.

PubMed

Wang, Guihua; Sheng, Lichao; Zhao, Dan; Sheng, Jiandong; Wang, Xiurong; Liao, Hong

2016-01-01

Soybean/maize intercropping has remarkable advantages in increasing crop yield and nitrogen (N) efficiency. However, little is known about the contributions of rhizobia or arbuscular mycorrhizal fungi (AMF) to yield increases and N acquisition in the intercropping system. Plus, the mechanisms controlling carbon (C) and N allocation in intercropping systems remain unsettled. In the present study, a greenhouse experiment combined with 15 N and 13 C labeling was conducted using various inoculation and nutrient treatments. The results showed that co-inoculation with AMF and rhizobia dramatically increased biomass and N content of soybean and maize, and moderate application of N and phosphorus largely amplified the effect of co-inoculation. Maize had a competitive advantage over soybean only under co-inoculation and moderate nutrient availability conditions, indicating that the effects of AMF and rhizobia in intercropping systems are closely related to nutrient status. Results from 15 N labeling showed that the amount of N transferred from soybean to maize in co-inoculations was 54% higher than that with AMF inoculation alone, with this increased N transfer partly resulting from symbiotic N fixation. The results from 13 C labeling showed that 13 C content increased in maize shoots and decreased in soybean roots with AMF inoculation compared to uninoculated controls. Yet, with co-inoculation, 13 C content increased in soybean. These results indicate that photosynthate assimilation is stimulated by AM symbiosis in maize and rhizobial symbiosis in soybean, but AMF inoculation leads to soybean investing more carbon than maize into common mycorrhizal networks (CMNs). Overall, the results herein demonstrate that the growth advantage of maize when intercropped with soybean is due to acquisition of N by maize via CMNs while this crop contributes less C into CMNs than soybean under co-inoculation conditions.

The metabolic fate of doubly stable isotope labelled heat-killed Lactobacillus johnsonii in humans.

PubMed

Wutzke, K D; Berg, D; Haffner, D

2008-02-01

In this study, heat-killed Lactobacillus johnsonii (La1), doubly labelled with (13)C and (15)N (hk-dlLa1), was used to follow the metabolic fate after oral administration in humans. Experimental study. University of Rostock, Children's Hospital, Research Laboratory. Ten healthy adults aged 23-26 years. The subjects received 74.6 mg/kg body weight hk-dlLa1 and 10 g alpha-D-raffinose together with breakfast. A sample of venous blood was taken after 2 h. Expired air samples were taken over 14 h, whereas urine and faeces were collected over a period of 48 h. (13)C- and (15)N-enrichments were measured by isotope ratio mass spectrometry. Hydrogen concentrations were measured by electrochemical detection. The orocaecal transit time (OCTT) was reached after 3.4 h. After 2 h, (13)C- and (15)N-enrichment of fibrinogen amounted to 2 and 25 p.p.m. excess, respectively. The (13)CO(2)-exhalation amounted to 9.2% of the ingested dose. The urinary excretion of (13)C and (15)N was 2.1 and 10.4% of the ingested dose, respectively, whereas the faecal excretion was 47.9 and 43.7% of the ingested dose, respectively. In comparison to OCTT of 3.4 h, both stable isotopes appear after 30 min in breath and urine, indicating that hk-dlLa1 is rapidly digested in the small bowel before reaching the caecum. This is confirmed by (13)C-and (15)N-enrichments of blood plasma fractions. The ingestion of hk-dlLa1 led to a (13)C- and (15)N-excretion of 59.2 and 54.1% of the ingested dose, respectively, of both stable isotopes.

Root-derived carbon and nitrogen from beech and ash trees differentially fuel soil animal food webs of deciduous forests.

PubMed

Zieger, Sarah L; Ammerschubert, Silke; Polle, Andrea; Scheu, Stefan

2017-01-01

Evidence is increasing that soil animal food webs are fueled by root-derived carbon (C) and also by root-derived nitrogen (N). Functioning as link between the above- and belowground system, trees and their species identity are important drivers structuring soil animal communities. A pulse labeling experiment using 15N and 13C was conducted by exposing beech (Fagus sylvatica) and ash (Fraxinus excelsior) seedlings to 13CO2 enriched atmosphere and tree leaves to 15N ammonium chloride solution in a plant growth chamber under controlled conditions for 72 h. C and N fluxes into the soil animal food web of beech, associated with ectomycorrhizal fungi (EMF), and ash, associated with arbuscular mycorrhizal fungi (AMF), were investigated at two sampling dates (5 and 20 days after labeling). All of the soil animal taxa studied incorporated root-derived C, while root-derived N was only incorporated into certain taxa. Tree species identity strongly affected C and N incorporation with the incorporation in the beech rhizosphere generally exceeding that in the ash rhizosphere. Incorporation differed little between 5 and 20 days after labeling indicating that both C and N are incorporated quickly into soil animals and are used for tissue formation. Our results suggest that energy and nutrient fluxes in soil food webs depend on the identity of tree species with the differences being associated with different types of mycorrhiza. Further research is needed to prove the generality of these findings and to quantify the flux of plant C and N into soil food webs of forests and other terrestrial ecosystems.

Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases.

PubMed

Liu, Jia-Ming; Huang, Xiao-Mei; Liu, Zhen-Bo; Lin, Shao-Qin; Li, Fei-Ming; Gao, Fei; Li, Zhi-Ming; Zeng, Li-Qing; Li, Lian-Ying; Ouyang, Ying

2009-08-26

A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)(n)-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)(n)-DMA complex containing several FITC molecules. F-ol-(FITC)(n)-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)(n)-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot(-1) for F-ol and 0.097 ag AP spot(-1) for FITC in F-ol-(FITC)(n)-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot(-1) for F-ol-DMA and 0.22 ag AP spot(-1) for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)(n)-DMA labelling of WGA was discussed.

Assessing Microbial Activity in Marcellus Shale Hydraulic Fracturing Fluids

NASA Astrophysics Data System (ADS)

Wishart, J. R.; Morono, Y.; Itoh, M.; Ijiri, A.; Hoshino, T.; Inagaki, F.; Verba, C.; Torres, M. E.; Colwell, F. S.

2014-12-01

Hydraulic fracturing (HF) produces millions of gallons of waste fluid which contains a microbial community adapted to harsh conditions such as high temperatures, high salinities and the presence of heavy metals and radionuclides. Here we present evidence for microbial activity in HF production fluids. Fluids collected from a Marcellus shale HF well were supplemented with 13C-labeled carbon sources and 15N-labeled ammonium at 25°C under aerobic or anaerobic conditions. Samples were analyzed for 13C and 15N incorporation at sub-micrometer scale by ion imaging with the JAMSTEC NanoSIMS to determine percent carbon and nitrogen assimilation in individual cells. Headspace CO2 and CH4 were analyzed for 13C enrichment using irm-GC/MS. At 32 days incubation carbon assimilation was observed in samples containing 1 mM 13C-labeled glucose under aerobic and anaerobic conditions with a maximum of 10.4 and 6.5% total carbon, respectively. Nitrogen assimilation of 15N ammonium observed in these samples were 0.3 and 0.8% of total nitrogen, respectively. Head space gas analysis showed 13C enrichment in CH4 in anaerobic samples incubated with 1mM 13C-labeled bicarbonate (2227 ‰) or methanol (98943 ‰). Lesser 13C enrichment of CO2 was observed in anaerobic samples containing 1 mM 13C-labeled acetate (13.7 ‰), methanol (29.9 ‰) or glucose (85.4 ‰). These results indicate metabolic activity and diversity in microbial communities present in HF flowback fluids. The assimilation of 13C-labeled glucose demonstrates the production of biomass, a critical part of cell replication. The production of 13CO2 and 13CH4 demonstrate microbial metabolism in the forms of respiration and methanogenesis, respectively. Methanogenesis additionally indicates the presence of an active archaeal community. This research shows that HF production fluid chemistry does not entirely inhibit microbial activity or growth and encourages further research regarding biogeochemical processes occurring in Marcellus shale HF wells. Biogeochemical activity may impact the efficacy of HF and natural gas production as well as the chemistry of produced fluids which have become an environmental and public health concern.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

PubMed Central

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

2016-01-01

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

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

NASA Astrophysics Data System (ADS)

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

2013-04-01

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

Flux Analysis of Free Amino Sugars and Amino Acids in Soils by Isotope Tracing with a Novel Liquid Chromatography/High Resolution Mass Spectrometry Platform

PubMed Central

2017-01-01

Soil fluxomics analysis can provide pivotal information for understanding soil biochemical pathways and their regulation, but direct measurement methods are rare. Here, we describe an approach to measure soil extracellular metabolite (amino sugar and amino acid) concentrations and fluxes based on a 15N isotope pool dilution technique via liquid chromatography and high-resolution mass spectrometry. We produced commercially unavailable 15N and 13C labeled amino sugars and amino acids by hydrolyzing peptidoglycan isolated from isotopically labeled bacterial biomass and used them as tracers (15N) and internal standards (13C). High-resolution (Orbitrap Exactive) MS with a resolution of 50 000 allowed us to separate different stable isotope labeled analogues across a large range of metabolites. The utilization of 13C internal standards greatly improved the accuracy and reliability of absolute quantification. We successfully applied this method to two types of soils and quantified the extracellular gross fluxes of 2 amino sugars, 18 amino acids, and 4 amino acid enantiomers. Compared to the influx and efflux rates of most amino acids, similar ones were found for glucosamine, indicating that this amino sugar is released through peptidoglycan and chitin decomposition and serves as an important nitrogen source for soil microorganisms. d-Alanine and d-glutamic acid derived from peptidoglycan decomposition exhibited similar turnover rates as their l-enantiomers. This novel approach offers new strategies to advance our understanding of the production and transformation pathways of soil organic N metabolites, including the unknown contributions of peptidoglycan and chitin decomposition to soil organic N cycling. PMID:28776982

Movement of nitrate fertilizer to glacial till and runoff from a claypan soil

USGS Publications Warehouse

Blevins, D.W.; Wilkison, D.H.; Kelly, B.P.; Silva, S.R.

1996-01-01

Although water from 20 to 25% of shallow farmstead wells in northern Missouri has concentrations of nitrate (NO3/-) exceeding 10 mg L-1 as nitrogen (N), many potential sources for this NO3/- are usually present. A field experiment was designed to trace and isolate the amount of a single application of N fertilizer lost to a glacial-till aquifer and runoff from a 400 m2 corn (Zea mays L.) plot with bromide (Br-) and isotopically labeled (15N) fertilizer. Soil at the plot is a Albaquic Hapludalf of the Adco Series containing a 61 cm claypan beneath 41 to 43 cm of topsoil. Groundwater levels ranged from 0.38 to 2.40 m below the land surface. Transport of water and NO3/- to the saturated zone was not substantially retarded by the claypan. Labeled-N fertilizer accounted for as much as 8.6 mg L-1 of the NO3/- (as N) in groundwater, but only in the top 1 to 2 m of the saturated zone. After two growing seasons (16 mo), <2% of the labeled-N fertilizer was lost to runoff, about 30% was in the saturated zone, 27.3% was removed with the grain, and about 5% remained in the unsaturated zone. A large part of the remaining labeled N may have been lost in gaseous N forms. The presence of labeled NO3/- only in the top 2 m of the aquifer, slow horizontal transport, and winter recharge indicate grass crops such as wheat (Triticum aestivum L.) or rye (Secale cereale L.) might be used to extract near- surface N during the winter recharge period. Also, fall fertilizations can be expected to readily leach. Because groundwater concentrations of labeled NO3/- were still increasing after two growing seasons, rotation of crops requiring small N inputs could be expected to limit the cumulative effect of large annual fertilizer applications on groundwater.

15N and13C NMR investigation of hydroxylamine-derivatized humic substances

USGS Publications Warehouse

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

1992-01-01

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

MYB Labeling by Immunohistochemistry Is More Sensitive and Specific for Breast Adenoid Cystic Carcinoma than MYB Labeling by FISH.

PubMed

Poling, Justin S; Yonescu, Raluca; Subhawong, Andrea P; Sharma, Rajni; Argani, Pedram; Ning, Yi; Cimino-Mathews, Ashley

2017-07-01

Breast adenoid cystic carcinoma (ACC) is a primary breast carcinoma that, like salivary gland ACC, displays the t(6;9) translocation resulting in the MYB-NFIB gene fusion and immunopositivity for MYB by immunohistochemistry (IHC). However, it is not well established whether MYB immunoreactivity or rearrangement can be used to support a diagnosis of ACC in a malignant basaloid or benign cribriform breast lesion. Whole sections of primary breast ACC (n=11), collagenous spherulosis (CS; n=7), and microglandular adenosis (MGA; n=5) and tissue microarrays containing 16 basal-like, triple-negative breast carcinomas (TNBC) were labeled for MYB by IHC and underwent MYB fluorescence in situ hybridization using a break-apart probe. Strong, diffuse nuclear MYB labeling was seen in 100% ACC compared with no cases of basal-like TNBC, CS, or MGA (P=0.0001). Any degree of nuclear MYB labeling was seen in 100% ACC compared with 54% of all other cases (P=0.007), with any labeling seen in 71% CS, 63% basal-like TNBC, and 0% MGA. MYB rearrangement was detected in 89% (8/9) of evaluable ACC compared with 4% (1/26) of all other evaluable cases (P=0.0001), with a rearrangement detected in 1 (7%; n=1/15) evaluable basal-like TNBC. Strong, diffuse nuclear labeling for MYB is more sensitive than MYB fluorescence in situ hybridization for breast ACC and can be used to support a diagnosis of ACC in a cribriform or basaloid lesion in the breast. However, weak and focal labeling should be interpreted with caution as it can be seen in other benign cribriform and malignant basaloid lesions.

Quality of anthelminthic medicines available in Jimma Ethiopia.

PubMed

Belew, Sileshi; Suleman, Sultan; Wynendaele, Evelien; D'Hondt, Matthias; Kosgei, Anne; Duchateau, Luc; De Spiegeleer, Bart

2018-01-01

Soil-transmitted helminthiasis and schistosomiasis are major public health problems in Ethiopia. Mass deworming of at-risk population using a single dose administration of 400mg albendazole (ABZ) or 500mg mebendazole (MBZ) for treatment of common intestinal worms and 40mg of praziquantel (PZQ) per kg body weight for treatment of schistosomiasis is one of the strategies recommended by World Health Organization (WHO) in order to control the morbidity of soil-transmitted helminthiasis and schistosomiasis. Since storage condition, climate, way of transportation and distribution route could all affect the quality of medicines, regular assessment by surveys is very critical to ensure the therapeutic outcome, to minimize risk of toxicity to the patient and resistance of parasites. Therefore, this study was conducted to assess the pharmaceutical quality of ABZ, MBZ and PZQ tablet brands commonly available in Jimma town (south west Ethiopia). Retail pharmacies (n=10) operating in Jimma town were selected using simple random sampling method. Samples of anthelminthic medicines available in the selected pharmacies were collected. Sample information was recorded and encompassed trade name, active ingredient name, manufacturer's name and full address, labeled medicine strength, dosage form, number of units per container, dosage statement, batch/lot number, manufacturing and expiry dates, storage information and presence of leaflets/package insert. Moreover, a first visual inspection was performed encompassing uniformity of color, uniformity of size, breaks, cracks, splits, embedded surface spots or visual contaminations. Finally, physico-chemical quality attributes investigated encompassed mass uniformity, quantity of active pharmaceutical ingredient (API), disintegration and dissolution, all following Pharmacopoeial test methods The physical characteristics of dosage form, packaging and labeling information of all samples complied with criteria given in the WHO checklists. The mass uniformity of tablets of each brand of ABZ, MBZ and PZQ complied with the pharmacopoeial specification limits, i.e no more than 2 individual masses >5% of average tablet weight, and none deviate by more than 10%. The quantity of APIs in all investigated tablet brands were within the 90-110% label claim (l.c.) limits, ranging between 95.05 and 110.09% l.c. Disintegration times were in line with the pharmacopoeial specification limit for immediate release (IR) tablets, ranging between 0.5 and 13min. However, the dissolution results (mean±SD, n=6) of one ABZ brand (i.e. Wormin ® , Q=59.21±0.99% at 30min) and two PZQ brands (i.e. Bermoxel ® , Q=63.43%±0.7 and Distocide ® , Q=62.43%±1.67, at 75min) showed poor dissolution, failing the United States Pharmacopoeia (USP) dissolution specification limit. Copyright © 2017 Elsevier B.V. All rights reserved.

Long-term fate of nitrate fertilizer in agricultural soils.

PubMed

Sebilo, Mathieu; Mayer, Bernhard; Nicolardot, Bernard; Pinay, Gilles; Mariotti, André

2013-11-05

Increasing diffuse nitrate loading of surface waters and groundwater has emerged as a major problem in many agricultural areas of the world, resulting in contamination of drinking water resources in aquifers as well as eutrophication of freshwaters and coastal marine ecosystems. Although empirical correlations between application rates of N fertilizers to agricultural soils and nitrate contamination of adjacent hydrological systems have been demonstrated, the transit times of fertilizer N in the pedosphere-hydrosphere system are poorly understood. We investigated the fate of isotopically labeled nitrogen fertilizers in a three-decade-long in situ tracer experiment that quantified not only fertilizer N uptake by plants and retention in soils, but also determined to which extent and over which time periods fertilizer N stored in soil organic matter is rereleased for either uptake in crops or export into the hydrosphere. We found that 61-65% of the applied fertilizers N were taken up by plants, whereas 12-15% of the labeled fertilizer N were still residing in the soil organic matter more than a quarter century after tracer application. Between 8-12% of the applied fertilizer had leaked toward the hydrosphere during the 30-y observation period. We predict that additional exports of (15)N-labeled nitrate from the tracer application in 1982 toward the hydrosphere will continue for at least another five decades. Therefore, attempts to reduce agricultural nitrate contamination of aquatic systems must consider the long-term legacy of past applications of synthetic fertilizers in agricultural systems and the nitrogen retention capacity of agricultural soils.

Long-term fate of nitrate fertilizer in agricultural soils

PubMed Central

Sebilo, Mathieu; Mayer, Bernhard; Nicolardot, Bernard; Pinay, Gilles; Mariotti, André

2013-01-01

Increasing diffuse nitrate loading of surface waters and groundwater has emerged as a major problem in many agricultural areas of the world, resulting in contamination of drinking water resources in aquifers as well as eutrophication of freshwaters and coastal marine ecosystems. Although empirical correlations between application rates of N fertilizers to agricultural soils and nitrate contamination of adjacent hydrological systems have been demonstrated, the transit times of fertilizer N in the pedosphere–hydrosphere system are poorly understood. We investigated the fate of isotopically labeled nitrogen fertilizers in a three–decade-long in situ tracer experiment that quantified not only fertilizer N uptake by plants and retention in soils, but also determined to which extent and over which time periods fertilizer N stored in soil organic matter is rereleased for either uptake in crops or export into the hydrosphere. We found that 61–65% of the applied fertilizers N were taken up by plants, whereas 12–15% of the labeled fertilizer N were still residing in the soil organic matter more than a quarter century after tracer application. Between 8–12% of the applied fertilizer had leaked toward the hydrosphere during the 30-y observation period. We predict that additional exports of 15N-labeled nitrate from the tracer application in 1982 toward the hydrosphere will continue for at least another five decades. Therefore, attempts to reduce agricultural nitrate contamination of aquatic systems must consider the long-term legacy of past applications of synthetic fertilizers in agricultural systems and the nitrogen retention capacity of agricultural soils. PMID:24145428

Synthesis and formation mechanistic investigation of nitrogen-doped carbon dots with high quantum yields and yellowish-green fluorescence

NASA Astrophysics Data System (ADS)

Hou, Juan; Wang, Wei; Zhou, Tianyu; Wang, Bo; Li, Huiyu; Ding, Lan

2016-05-01

Heteroatom doped carbon dots (CDs) have received increasing attention due to their unique properties and related applications. However, previously reported CDs generally show strong emission only in the blue-light region, thus restricting their further applications. And the fundamental investigation on the preparation process is always neglected. Herein, we have developed a simple and solvent-free synthetic strategy to fabricate nitrogen-doped CDs (N-CDs) from citric acid and dicyandiamide. The as-prepared N-CDs exhibited a uniform size distribution, strong yellowish-green fluorescence emission and a high quantum yield of 73.2%. The products obtained at different formation stages were detailedly characterized by transmission electron microscopy, X-ray diffraction spectrometer, X-ray photoelectron spectroscopy and UV absorbance spectroscopy. A possible formation mechanism has thus been proposed including dehydration, polymerization and carbonization. Furthermore, the N-CDs could serve as a facile and label-free probe for the detection of iron and fluorine ions with detection limits of 50 nmol L-1 and 75 nmol L-1, respectively.Heteroatom doped carbon dots (CDs) have received increasing attention due to their unique properties and related applications. However, previously reported CDs generally show strong emission only in the blue-light region, thus restricting their further applications. And the fundamental investigation on the preparation process is always neglected. Herein, we have developed a simple and solvent-free synthetic strategy to fabricate nitrogen-doped CDs (N-CDs) from citric acid and dicyandiamide. The as-prepared N-CDs exhibited a uniform size distribution, strong yellowish-green fluorescence emission and a high quantum yield of 73.2%. The products obtained at different formation stages were detailedly characterized by transmission electron microscopy, X-ray diffraction spectrometer, X-ray photoelectron spectroscopy and UV absorbance spectroscopy. A possible formation mechanism has thus been proposed including dehydration, polymerization and carbonization. Furthermore, the N-CDs could serve as a facile and label-free probe for the detection of iron and fluorine ions with detection limits of 50 nmol L-1 and 75 nmol L-1, respectively. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02701f

Nuclear magnetic resonance (NMR) studies on the biosynthesis of fusaric acid from Fusarium oxysporum f. sp. vasinfectum.

PubMed

Stipanovic, Robert D; Wheeler, Michael H; Puckhaber, Lorraine S; Liu, Jinggao; Bell, Alois A; Williams, Howard J

2011-05-25

Fusarium oxysporum is a fungal pathogen that attacks many important plants. Uniquely pathogenic strains of F. oxysporum f. sp. vasinfectum were inadvertently imported into the United States on live cottonseed for dairy cattle feed. These strains produce exceptionally high concentrations of the phytotoxin fusaric acid. Thus, fusaric acid may be a critical component in the pathogenicity of these biotypes. This study investigated the biosynthesis of fusaric acid using (13)C-labeled substrates including [1,2-(13)C(2)]acetate as well as (13)C- and (15)N-labeled aspartate and [(15)N]glutamine. The incorporation of labeled substrates is consistent with the biosynthesis of fusaric acid from three acetate units at C5-C6, C7-C8, and C9-C10, with the remaining carbons being derived from aspartate via oxaloacetate and the TCA cycle; the oxaloacetate originates in part by transamination of aspartate, but most of the oxaloacetate is derived by deamination of aspartate to fumarate by aspartase. The nitrogen from glutamine is more readily incorporated into fusaric acid than that from aspartate.

Titanium Dioxide Nanoparticles (TiO₂) Quenching Based Aptasensing Platform: Application to Ochratoxin A Detection.

PubMed

Sharma, Atul; Hayat, Akhtar; Mishra, Rupesh K; Catanante, Gaëlle; Bhand, Sunil; Marty, Jean Louis

2015-09-22

We demonstrate for the first time, the development of titanium dioxide nanoparticles (TiO₂) quenching based aptasensing platform for detection of target molecules. TiO₂ quench the fluorescence of FAM-labeled aptamer (fluorescein labeled aptamer) upon the non-covalent adsorption of fluorescent labeled aptamer on TiO₂ surface. When OTA interacts with the aptamer, it induced aptamer G-quadruplex complex formation, weakens the interaction between FAM-labeled aptamer and TiO₂, resulting in fluorescence recovery. As a proof of concept, an assay was employed for detection of Ochratoxin A (OTA). At optimized experimental condition, the obtained limit of detection (LOD) was 1.5 nM with a good linearity in the range 1.5 nM to 1.0 µM for OTA. The obtained results showed the high selectivity of assay towards OTA without interference to structurally similar analogue Ochratoxin B (OTB). The developed aptamer assay was evaluated for detection of OTA in beer sample and recoveries were recorded in the range from 94.30%-99.20%. Analytical figures of the merits of the developed aptasensing platform confirmed its applicability to real samples analysis. However, this is a generic aptasensing platform and can be extended for detection of other toxins or target analyte.

Probing the Carbonyl Functionality of a Petroleum Resin and Asphaltene through Oximation and Schiff Base Formation in Conjunction with N-15 NMR

PubMed Central

Thorn, Kevin A.; Cox, Larry G.

2015-01-01

Despite recent advances in spectroscopic techniques, there is uncertainty regarding the nature of the carbonyl groups in the asphaltene and resin fractions of crude oil, information necessary for an understanding of the physical properties and environmental fate of these materials. Carbonyl and hydroxyl group functionalities are not observed in natural abundance 13C nuclear magnetic resonance (NMR) spectra of asphaltenes and resins and therefore require spin labeling techniques for detection. In this study, the carbonyl functionalities of the resin and asphaltene fractions from a light aliphatic crude oil that is the source of groundwater contamination at the long term USGS study site near Bemidji, Minnesota, have been examined through reaction with 15N-labeled hydroxylamine and aniline in conjunction with analysis by solid and liquid state 15N NMR. Ketone groups were revealed through 15N NMR detection of their oxime and Schiff base derivatives, and esters through their hydroxamic acid derivatives. Anilinohydroquinone adducts provided evidence for quinones. Some possible configurations of the ketone groups in the resin and asphaltene fractions can be inferred from a consideration of the likely reactions that lead to heterocyclic condensation products with aniline and to the Beckmann reaction products from the initially formed oximes. These include aromatic ketones and ketones adjacent to quaternary carbon centers, β-hydroxyketones, β-diketones, and β-ketoesters. In a solid state cross polarization/magic angle spinning (CP/MAS) 15N NMR spectrum recorded on the underivatized asphaltene as a control, carbazole and pyrrole-like nitrogens were the major naturally abundant nitrogens detected. PMID:26556054

Label-Free Alignment of Nonmagnetic Particles in a Small Uniform Magnetic Field.

PubMed

Wang, Zhaomeng; Wang, Ying; Wu, Rui Ge; Wang, Z P; Ramanujan, R V

2018-01-01

Label-free manipulation of biological entities can minimize damage, increase viability and improve efficiency of subsequent analysis. Understanding the mechanism of interaction between magnetic and nonmagnetic particles in an inverse ferrofluid can provide a mechanism of label-free manipulation of such entities in a uniform magnetic field. The magnetic force, induced by relative magnetic susceptibility difference between nonmagnetic particles and surrounding magnetic particles as well as particle-particle interaction were studied. Label-free alignment of nonmagnetic particles can be achieved by higher magnetic field strength (Ba), smaller particle spacing (R), larger particle size (rp1), and higher relative magnetic permeability difference between particle and the surrounding fluid (Rμr). Rμr can be used to predict the direction of the magnetic force between both magnetic and nonmagnetic particles. A sandwich structure, containing alternate layers of magnetic and nonmagnetic particle chains, was studied. This work can be used for manipulation of nonmagnetic particles in lab-on-a-chip applications.

Determination of radionuclides and radiochemical impurities produced by in-house cyclotron irradiation and subsequent radiosynthesis of PET tracers.

PubMed

Ishiwata, Kiichi; Hayashi, Kunpei; Sakai, Masanari; Kawauchi, Sugio; Hasegawa, Hideaki; Toyohara, Jun

2017-01-01

To elucidate the radionuclides and radiochemical impurities included in radiosynthesis processes of positron emission tomography (PET) tracers. Target materials and PET tracers were produced using a cyclotron/synthesis system from Sumitomo Heavy Industry. Positron and γ-ray emitting radionuclides were quantified by measuring radioactivity decay and using the high-purity Ge detector, respectively. Radiochemical species in gaseous and aqueous target materials were analyzed by gas and ion chromatography, respectively. Target materials had considerable levels of several positron emitters in addition to the positron of interest, and in the case of aqueous target materials extremely low levels of many γ-emitters. Five 11 C-, 15 O-, or 18 F-labeled tracers produced from gaseous materials via chemical reactions had no radionuclidic impurities, whereas 18 F-FDG, 18 F-NaF, and 13 N-NH 3 produced from aqueous materials had several γ-emitters as well as impure positron emitters. 15 O-Labeled CO 2 , O 2 , and CO had a radionuclidic impurity 13 N-N 2 (0.5-0.7 %). Target materials had several positron emitters other than the positron of interest, and extremely low level γ-emitters in the case of aqueous materials. PET tracers produced from gaseous materials except for 15 O-labeled gases had no impure radionuclides, whereas those derived from aqueous materials contained acceptable levels of impure positron emitters and extremely low levels of several γ-emitters.

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

PubMed Central

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

2012-01-01

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

Orientation and Order of the Amide Group of Sphingomyelin in Bilayers Determined by Solid-State NMR

PubMed Central

Matsumori, Nobuaki; Yamaguchi, Toshiyuki; Maeta, Yoshiko; Murata, Michio

2015-01-01

Sphingomyelin (SM) and cholesterol (Chol) are considered essential for the formation of lipid rafts; however, the types of molecular interactions involved in this process, such as intermolecular hydrogen bonding, are not well understood. Since, unlike other phospholipids, SM is characterized by the presence of an amide group, it is essential to determine the orientation of the amide and its order in the lipid bilayers to understand the nature of the hydrogen bonds in lipid rafts. For this study, 1′-13C-2-15N-labeled and 2′-13C-2-15N-labeled SMs were prepared, and the rotational-axis direction and order parameters of the SM amide in bilayers were determined based on 13C and 15N chemical-shift anisotropies and intramolecular 13C-15N dipole coupling constants. Results revealed that the amide orientation was minimally affected by Chol, whereas the order was enhanced significantly in its presence. Thus, Chol likely promotes the formation of an intermolecular hydrogen-bond network involving the SM amide without significantly changing its orientation, providing a higher order to the SM amide. To our knowledge, this study offers new insight into the significance of the SM amide orientation with regard to molecular recognition in lipid rafts, and therefore provides a deeper understanding of the mechanism of their formation. PMID:26083921

Magnetic resonance imaging of single co-labeled mesenchymal stromal cells after intracardial injection in mice.

PubMed

Salamon, J; Wicklein, D; Didié, M; Lange, C; Schumacher, U; Adam, G; Peldschus, K

2014-04-01

The aim of this study was to establish co-labeling of mesenchymal stromal cells (MSC) for the detection of single MSC in-vivo by MRI and histological validation. Mouse MSC were co-labeled with fluorescent iron oxide micro-particles and carboxyfluorescein succinimidyl ester (CFSE). The cellular iron content was determined by atomic absorption spectrometry. Cell proliferation and expression of characteristic surface markers were determined by flow cytometry. The chondrogenic differentiation capacity was assessed. Different amounts of cells (n1 = 5000, n2 = 15 000, n3 = 50 000) were injected into the left heart ventricle of 12 mice. The animals underwent sequential MRI on a clinical 3.0 T scanner (Intera, Philips Medical Systems, Best, The Netherlands). For histological validation cryosections were examined by fluorescent microscopy. Magnetic and fluorescent labeling of MSC was established (mean cellular iron content 23.6 ± 3 pg). Flow cytometry showed similar cell proliferation and receptor expression of labeled and unlabeled MSC. Chondrogenic differentiation of labeled MSC was verified. After cell injection MRI revealed multiple signal voids in the brain and fewer signal voids in the kidneys. In the brain, an average of 4.6 ± 1.2 (n1), 9.0 ± 3.6 (n2) and 25.0 ± 1.0 (n3) signal voids were detected per MRI slice. An average of 8.7 ± 3.1 (n1), 22.0 ± 6.1 (n2) and 89.8 ± 6.5 (n3) labeled cells per corresponding stack of adjacent cryosections could be detected in the brain. Statistical correlation of the numbers of MRI signal voids in the brain and single MSC found by histology revealed a correlation coefficient of r = 0.91. The study demonstrates efficient magnetic and fluorescent co-labeling of MSC and their detection on a single cell level in mice by in-vivo MRI and histology. The described techniques may broaden the methods for in-vivo tracking of MSC. • Detection of single magnetically labeled MSC in-vivo using a clinical 3.0 T MRI is possible.• Fluorescent and magnetic co-labeling does not affect cell vitality.• The number of cells detected by MRI and histology has a high correlation. © Georg Thieme Verlag KG Stuttgart · New York.

Modeling an in-register, parallel "iowa" aβ fibril structure using solid-state NMR data from labeled samples with rosetta.

PubMed

Sgourakis, Nikolaos G; Yau, Wai-Ming; Qiang, Wei

2015-01-06

Determining the structures of amyloid fibrils is an important first step toward understanding the molecular basis of neurodegenerative diseases. For β-amyloid (Aβ) fibrils, conventional solid-state NMR structure determination using uniform labeling is limited by extensive peak overlap. We describe the characterization of a distinct structural polymorph of Aβ using solid-state NMR, transmission electron microscopy (TEM), and Rosetta model building. First, the overall fibril arrangement is established using mass-per-length measurements from TEM. Then, the fibril backbone arrangement, stacking registry, and "steric zipper" core interactions are determined using a number of solid-state NMR techniques on sparsely (13)C-labeled samples. Finally, we perform Rosetta structure calculations with an explicitly symmetric representation of the system. We demonstrate the power of the hybrid Rosetta/NMR approach by modeling the in-register, parallel "Iowa" mutant (D23N) at high resolution (1.2Å backbone rmsd). The final models are validated using an independent set of NMR experiments that confirm key features. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Analysis of hydrazine in drinking water by isotope dilution gas chromatography/tandem mass spectrometry with derivatization and liquid-liquid extraction.

PubMed

Davis, William E; Li, Yongtao

2008-07-15

A new isotope dilution gas chromatography/chemical ionization/tandem mass spectrometric method was developed for the analysis of carcinogenic hydrazine in drinking water. The sample preparation was performed by using the optimized derivatization and multiple liquid-liquid extraction techniques. Using the direct aqueous-phase derivatization with acetone, hydrazine and isotopically labeled hydrazine-(15)N2 used as the surrogate standard formed acetone azine and acetone azine-(15)N2, respectively. These derivatives were then extracted with dichloromethane. Prior to analysis using methanol as the chemical ionization reagent gas, the extract was dried with anhydrous sodium sulfate, concentrated through evaporation, and then fortified with isotopically labeled N-nitrosodimethylamine-d6 used as the internal standard to quantify the extracted acetone azine-(15)N2. The extracted acetone azine was quantified against the extracted acetone azine-(15)N2. The isotope dilution standard calibration curve resulted in a linear regression correlation coefficient (R) of 0.999. The obtained method detection limit was 0.70 ng/L for hydrazine in reagent water samples, fortified at a concentration of 1.0 ng/L. For reagent water samples fortified at a concentration of 20.0 ng/L, the mean recoveries were 102% with a relative standard deviation of 13.7% for hydrazine and 106% with a relative standard deviation of 12.5% for hydrazine-(15)N2. Hydrazine at 0.5-2.6 ng/L was detected in 7 out of 13 chloraminated drinking water samples but was not detected in the rest of the chloraminated drinking water samples and the studied chlorinated drinking water sample.

Nitrogen metabolism in Lignifying Pinus taeda cell cultures

NASA Technical Reports Server (NTRS)

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

1996-01-01

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

A traffic light food labeling intervention increases consumer awareness of health and healthy choices at the point-of-purchase

PubMed Central

Sonnenberg, Lillian; Gelsomin, Emily; Levy, Douglas E.; Riis, Jason; Barraclough, Susan; Thorndike, Anne N.

2014-01-01

Objective We surveyed customers in a hospital cafeteria in Boston, Massachusetts before and after implementation of traffic light food labeling to determine the effect of labels on customers’ awareness and purchase of healthy foods. Methods Cafeteria items were identified as red (unhealthy), yellow (less healthy), or green (healthy). Customers were interviewed before (N = 166) and after (N = 223) labeling was implemented. Each respondent was linked to cash register data to determine the proportion of red, yellow, and green items purchased. Data were collected from February–April 2010. We compared responses to survey questions and mean proportion of red, yellow, and green items per transaction between customers interviewed during baseline and customers interviewed during the intervention. Survey response rate was 60%. Results Comparing responses during labeling intervention to baseline, more respondents identified health/ nutrition as an important factor in their purchase (61% vs. 46%, p = 0.004) and reported looking at nutrition information (33% vs. 15%, p < 0.001). Respondents who noticed labels during the intervention and reported that labels influenced their purchases were more likely to purchase healthier items than respondents who did not notice labels (p < 0.001 for both). Conclusion Traffic light food labels prompted individuals to consider their health and to make healthier choices at point-of-purchase. PMID:23859926

NMR scalar couplings across Watson–Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy

PubMed Central

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

1998-01-01

This paper describes the NMR observation of 15N—15N and 1H—15N scalar couplings across the hydrogen bonds in Watson–Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6–7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2–3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids. PMID:9826668

Root-derived carbon and nitrogen from beech and ash trees differentially fuel soil animal food webs of deciduous forests

PubMed Central

Ammerschubert, Silke; Polle, Andrea; Scheu, Stefan

2017-01-01

Evidence is increasing that soil animal food webs are fueled by root-derived carbon (C) and also by root-derived nitrogen (N). Functioning as link between the above- and belowground system, trees and their species identity are important drivers structuring soil animal communities. A pulse labeling experiment using 15N and 13C was conducted by exposing beech (Fagus sylvatica) and ash (Fraxinus excelsior) seedlings to 13CO2 enriched atmosphere and tree leaves to 15N ammonium chloride solution in a plant growth chamber under controlled conditions for 72 h. C and N fluxes into the soil animal food web of beech, associated with ectomycorrhizal fungi (EMF), and ash, associated with arbuscular mycorrhizal fungi (AMF), were investigated at two sampling dates (5 and 20 days after labeling). All of the soil animal taxa studied incorporated root-derived C, while root-derived N was only incorporated into certain taxa. Tree species identity strongly affected C and N incorporation with the incorporation in the beech rhizosphere generally exceeding that in the ash rhizosphere. Incorporation differed little between 5 and 20 days after labeling indicating that both C and N are incorporated quickly into soil animals and are used for tissue formation. Our results suggest that energy and nutrient fluxes in soil food webs depend on the identity of tree species with the differences being associated with different types of mycorrhiza. Further research is needed to prove the generality of these findings and to quantify the flux of plant C and N into soil food webs of forests and other terrestrial ecosystems. PMID:29236746

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

NASA Astrophysics Data System (ADS)

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

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

U.S. EPA, Pesticide Product Label, MICRO-TECH HERBICIDE, 12/15/1983

EPA Pesticide Factsheets

2011-04-14

... UII' ul!1lt'1 1 102. lII(.h{') 01 !lOll llc~ lor n".umum Of ... ncr"',,! d.:itQudlt' !'trlelmdn I' '. r:;"'I'~": "1l"n 1'Tl1)1\\lulf' I~ If'ctIWf'd "'thl~ 1 (loll' ""f" art"·· d!'nll .tn\\'! ...

How interacting fungal species and mineral nitrogen inputs affect transfer of nitrogen from litter via arbuscular mycorrhizal mycelium.

PubMed

He, Yuejun; Cornelissen, J Hans C; Zhong, Zhangcheng; Dong, Ming; Jiang, Changhong

2017-04-01

In the karst landscape, widespread in the world including southern China, soil nutrient supply is strongly constrained. In such environments, arbuscular mycorrhizal (AM) fungi may facilitate plant nutrient uptake. However, the possible role of different AM fungal species, and their interactions, especially in transferring nitrogen (N) from litter to plant, is poorly understood. We conducted two microcosm experiments to investigate the role that two karst soil AM fungi, Glomus etunicatum and Glomus mosseae, play in the transfer of N from decomposing litter to the host plant and to determine how N availability influences these processes. In experiment 1, Cinnamomum camphora tree seedlings were grown in compartments inoculated with G. etunicatum. Lolium perenne leaf litter labeled with δ 15 N was added to the soil in unplanted compartments. Compartments containing the δ 15 N labeled litter were either accessible to hyphae but not to seedling roots or were not accessible to hyphae or roots. The addition of mineral N to one of the host compartments at the start of the experiment significantly increased the biomass of the C. camphora seedlings, N content and N:P ratio, AM mycelium length, and soil microbial biomass carbon and N. However, significantly, more δ 15 N was acquired, from the leaf litter by the AM hyphae and transferred to the host when mineral N was not added to the soil. In experiment 2, in which C. camphora seedlings were inoculated with both G. etunicatum and G. mosseae rather than with G. mosseae alone, there was a significant increase in mycelial growth (50.21%), in soil microbial biomass carbon (417.73%) in the rhizosphere, and in the amount of δ 15 N that was transferred to the host. These findings suggest that maintaining AM fungal diversity in karst soils could be important for mediating N transfer from organic material to host plants in N-poor soils.

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

1989-01-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin.

PubMed

Hicks, G R; Rayle, D L; Jones, A M; Lomax, T L

1989-07-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

Biodegradation and mineralization of isotopically labeled TNT and RDX in anaerobic marine sediments.

PubMed

Ariyarathna, Thivanka; Vlahos, Penny; Smith, Richard W; Fallis, Stephen; Groshens, Thomas; Tobias, Craig

2017-05-01

The lack of knowledge on the fate of explosive compounds 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), particularly in marine ecosystems, constrains the application of bioremediation techniques in explosive-contaminated coastal sites. The authors present a comparative study on anaerobic biodegradation and mineralization of 15 N-nitro group isotopically labeled TNT and RDX in organic carbon-rich, fine-grained marine sediment with native microbial assemblages. Separate sediment slurry experiments were carried out for TNT and RDX at 23°C for 16 d. Dissolved and sediment-sorbed fractions of parent and transformation products, isotopic compositions of sediment, and mineralization products of the dissolved inorganic N pool ( 15 NH 4 + , 15 NO 3 - , 15 NO 2 - , and 15 N 2 ) were measured. The rate of TNT removal from the aqueous phase was faster (0.75 h -1 ) than that of RDX (0.37 h -1 ), and 15 N accumulation in sediment was higher in the TNT (13%) than the RDX (2%) microcosms. Mono-amino-dinitrotoluenes were identified as intermediate biodegradation products of TNT. Two percent of the total spiked TNT-N is mineralized to dissolved inorganic N through 2 different pathways: denitration as well as deamination and formation of NH 4 + , facilitated by iron and sulfate reducing bacteria in the sediments. The majority of the spiked TNT-N (85%) is in unidentified pools by day 16. Hexahydro-1,3,5-trinitro-1,3,5-triazine (10%) biodegrades to nitroso derivatives, whereas 13% of RDX-N in nitro groups is mineralized to dissolved inorganic N anaerobically by the end of the experiment. The primary identified mineralization end product of RDX (40%) is NH 4 + , generated through either deamination or mono-denitration, followed by ring breakdown. A reasonable production of N 2 gas (13%) was seen in the RDX system but not in the TNT system. Sixty-eight percent of the total spiked RDX-N is in an unidentified pool by day 16 and may include unquantified mineralization products dissolved in water. Environ Toxicol Chem 2017;36:1170-1180. © 2016 SETAC. © 2016 SETAC.

Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajagopal, P.; Klevit, R.E.

1994-12-01

HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residuemore » at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.« less

Cross-cultural measurement equivalence of the EQ-5D-5L items for English-speaking Asians in Singapore.

PubMed

Luo, N; Wang, Y; How, C H; Wong, K Y; Shen, L; Tay, E G; Thumboo, J; Herdman, M

2015-06-01

To investigate how the response labels of the 5-level EQ-5D (EQ-5D-5L) items are interpreted and used by English-speaking Chinese and non-Chinese Singaporeans, as a means to assessing whether those items are cross-culturally equivalent health-status measures in this Asian population. In face-to-face interviews, Chinese, Malay and Indian visitors to a primary care institution in Singapore were asked to rate the relative severity conveyed by EQ-5D-5L response labels, each containing the keyword of 'no(t),' 'slight(ly),' 'moderate(ly),' 'severe(ly),' or 'unable'/'extreme(ly),' using a 0-100 numerical rating scale. Participants were also asked to describe 25 hypothetical health states using the EQ-5D-5L response labels. Differences between Chinese and Malay/Indian participants in label interpretation and selection were examined using multivariate regression analysis to adjust for participant characteristics. The differences in adjusted mean severity scores for individual EQ-5D-5L labels between Chinese (n = 148) and non-Chinese (Malay: n = 53; Indian: n = 56) participants ranged from 0.0 to 9.0. The relative severity of the labels to the participants supported the ordinality of the EQ-5D-5L response labels and was similar across ethnic groups. Chinese and non-Chinese participants selected similar response labels to describe each hypothetical health state, with the adjusted odds ratios of selecting any type of the five response labels for non-Chinese versus Chinese participants ranging from 0.92 to 1.15 (p > 0.05 for all). The EQ-5D-5L items are likely to generate equivalent health outcomes between English-speaking Chinese and non-Chinese Singaporeans.

Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morris, D.I.; Robbins, J.D.; Ruoho, A.E.

1991-07-15

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolinmore » but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.« less

Factors associated with nutrition label use among female college students applying the theory of planned behavior.

PubMed

Lim, Hyun Jeong; Kim, Min Ju; Kim, Kyung Won

2015-02-01

Use of nutrition labels in food selection is recommended for consumers. The aim of this study is to examine factors, mainly beliefs explaining nutrition label use in female college students based on the Theory of Planned Behavior (TPB). The subjects were female college students from a university in Seoul, Korea. The survey questionnaire was composed of items examining general characteristics, nutrition label use, behavioral beliefs, normative beliefs, corresponding motivation to comply, and control beliefs. The subjects (n = 300) responded to the questionnaire by self-report, and data from 275 students were analyzed using t-test or χ(2)-test. The results showed that 37.8% of subjects were nutrition label users. Three out of 15 behavioral beliefs differed significantly by nutrition label use. Nutrition label users agreed more strongly on the benefits of using nutrition labels including 'comparing and selecting better foods' (P < 0.001), 'selecting healthy foods' (P < 0.05). The negative belief of 'annoying' was stronger in non-users than in users (P < 0.001). Three out of 7 sources (parents, siblings, best friend) were important in nutrition label use. Twelve out of 15 control beliefs differed significantly by nutrition label use. These included beliefs regarding constraints of using nutrition labels (e.g., time, spending money for healthy foods) and lack of nutrition knowledge (P < 0.001). Perceived confidence in understanding and applying the specifics of nutrition labels in food selection was also significantly related to nutrition label use (P < 0.001). This study found that the beliefs, especially control beliefs, suggested in the TPB were important in explaining nutrition label use. To promote nutrition label use, nutrition education might focus on increasing perceived control over constraints of using nutrition labels, acquiring skills for checking nutrition labels, as well as the benefits of using nutrition labels and receiving support from significant others for nutrition label use.

Factors associated with nutrition label use among female college students applying the theory of planned behavior

PubMed Central

Lim, Hyun Jeong; Kim, Min Ju

2015-01-01

BACKGROUND/OBJECTIVES Use of nutrition labels in food selection is recommended for consumers. The aim of this study is to examine factors, mainly beliefs explaining nutrition label use in female college students based on the Theory of Planned Behavior (TPB). SUBJECTS/METHODS The subjects were female college students from a university in Seoul, Korea. The survey questionnaire was composed of items examining general characteristics, nutrition label use, behavioral beliefs, normative beliefs, corresponding motivation to comply, and control beliefs. The subjects (n = 300) responded to the questionnaire by self-report, and data from 275 students were analyzed using t-test or χ2-test. RESULTS The results showed that 37.8% of subjects were nutrition label users. Three out of 15 behavioral beliefs differed significantly by nutrition label use. Nutrition label users agreed more strongly on the benefits of using nutrition labels including 'comparing and selecting better foods' (P < 0.001), 'selecting healthy foods' (P < 0.05). The negative belief of 'annoying' was stronger in non-users than in users (P < 0.001). Three out of 7 sources (parents, siblings, best friend) were important in nutrition label use. Twelve out of 15 control beliefs differed significantly by nutrition label use. These included beliefs regarding constraints of using nutrition labels (e.g., time, spending money for healthy foods) and lack of nutrition knowledge (P < 0.001). Perceived confidence in understanding and applying the specifics of nutrition labels in food selection was also significantly related to nutrition label use (P < 0.001). CONCLUSIONS This study found that the beliefs, especially control beliefs, suggested in the TPB were important in explaining nutrition label use. To promote nutrition label use, nutrition education might focus on increasing perceived control over constraints of using nutrition labels, acquiring skills for checking nutrition labels, as well as the benefits of using nutrition labels and receiving support from significant others for nutrition label use. PMID:25671070

Hyperpolarization of “Neat” Liquids by NMR Signal Amplification by Reversible Exchange

PubMed Central

2016-01-01

We report NMR Signal Amplification by Reversible Exchange (SABRE) hyperpolarization of the rare isotopes in “neat” liquids, each composed only of an otherwise pure target compound with isotopic natural abundance (n.a.) and millimolar concentrations of dissolved catalyst. Pyridine (Py) or Py derivatives are studied at 0.4% isotopic natural abundance 15N, deuterated, 15N enriched, and in various combinations using the SABRE-SHEATH variant (microTesla magnetic fields to permit direct 15N polarization from parahydrogen via reversible binding and exchange with an Ir catalyst). We find that the dilute n.a. 15N spin bath in Py still channels spin order from parahydrogen to dilute 15N spins, without polarization losses due to the presence of 14N or 2H. We demonstrate P15N ≈ 1% (a gain of 2900 fold relative to thermal polarization at 9.4 T) at high substrate concentrations. This fundamental finding has a significant practical benefit for screening potentially hyperpolarizable contrast agents without labeling. The capability of screening at n.a. level of 15N is demonstrated on examples of mono- and dimethyl-substituted Py (picolines and lutidines previously identified as promising pH sensors), showing that the presence of a methyl group in the ortho position significantly decreases SABRE hyperpolarization. PMID:26029349

A microfluidic device for continuous manipulation of biological cells using dielectrophoresis.

PubMed

Das, Debanjan; Biswas, Karabi; Das, Soumen

2014-06-01

The present study demonstrates the design, simulation, fabrication and testing of a label-free continuous manipulation and separation micro-device of particles/biological cells suspended on medium based on conventional dielectrophoresis. The current dielectrophoretic device uses three planner electrodes to generate non-uniform electric field and induces both p-DEP and n-DEP force simultaneously depending on the dielectric properties of the particles and thus influencing at least two types of particles at a time. Numerical simulations were performed to predict the distribution of non-uniform electric field, DEP force and particle trajectories. The device is fabricated utilizing the advantage of bonding between PDMS and SU8 polymer. The p-DEP particles move away from the center of the streamline, while the n-DEP particles will follow the central streamline along the channel length. Dielectrophoretic effects were initially tested using polystyrene beads followed by manipulation of HeLa cells. In the experiment, it was observed that polystyrene beads in DI water always response as n-DEP up to 1MHz frequency, whereas HeLa cells in PBS medium response as n-DEP up to 400kHz frequency and then it experiences p-DEP up to 1MHz. Further, the microscopic observations of DEP responses of HeLa cells were verified by performing trapping experiment at static condition. Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.

Use of Nitrogen Isotope To Determine Fertilizer- and Soil-Derived Ammonia Volatilization in a Rice/Wheat Rotation System.

PubMed

Zhao, Xu; Yan, Xiaoyuan; Xie, Yingxin; Wang, Shenqiang; Xing, Guangxi; Zhu, Zhaoliang

2016-04-20

The nitrogen (N) isotope method reveals that application of fertilizer N can increase crop uptake or denitrification and leaching losses of native soil N via the "added N interaction". However, there is currently little evidence of the impact of added N on soil N losses through NH3 volatilization using (15)N methodologies. In the present study, a three-year rice/wheat rotated experiment with 30% (15)N-labeled urea applied in the first rice season and unlabeled urea added in the following five crop seasons was performed to investigate volatilization of NH3 from fertilizer and soil N. We found 9.28% of NH3 loss from (15)N urea and 2.88-7.70% declines in (15)N-NH3 abundance occurred during the first rice season, whereas 0.11% of NH3 loss from (15)N urea and 0.02-0.21% enrichments in (15)N-NH3 abundance happened in the subsequent seasons. The contributions of fertilizer- and soil-derived N to NH3 volatilization from a rice/wheat rotation were 75.8-88.4 and 11.6-24.2%, respectively. These distinct variations in (15)N-NH3 and substantial soil-derived NH3 suggest that added N clearly interacts with the soil source contributing to NH3 volatilization.

Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by sup 15 N NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Dijk, A.A.; de Lange, L.C.M.; Robillard, G.T.

1990-09-04

The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with {sup 15}N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with {sup 15}N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the N{delta}1 and N{epsilon}2 positions or at only the N{delta}1 position. {sup 15}N NMR spectra of two synthesized model compound, phosphoimidazole and phosphomethylimidazole, were also recorded. Themore » authors show that, prior to phosphorylation, the protonated His15 N{epsilon}2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated N{delta}1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 N{delta}1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed.« less

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

USDA-ARS?s Scientific Manuscript database

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

Sulfogalactosylglycerolipid is involved in human gamete interaction.

PubMed

Weerachatyanukul, W; Rattanachaiyanont, M; Carmona, E; Furimsky, A; Mai, A; Shoushtarian, A; Sirichotiyakul, S; Ballakier, H; Leader, A; Tanphaichitr, N

2001-12-01

Recent results from our laboratory have revealed the role of sulfogalactosylglycerolipid (SGG) in mouse sperm-zona pellucida (ZP) binding. In this report, we demonstrated the presence of SGG in Percoll-gradient centrifuged (PGC) human sperm by high performance thin layer chromatography with orcinol and Azure A staining, specific for glycolipids and sulfolipids, respectively. SGG in human PGC sperm was quantified by its affinity to Azure A to be 12-15 mol% of sperm lipids. Indirect immunofluorescence revealed that SGG existed on both live and aldehyde fixed human sperm in the head region. Pretreatment of human PGC sperm with affinity purified antiSGG Fab markedly inhibited sperm binding to the ZP in a concentration dependent manner, without any changes in the spontaneous acrosome rate or sperm motility parameters. Fluorescently labeled SGG liposomes also bound uniformly to isolated human ZP, while fluorescently labeled galactosylglycerolipid (GG, SGG's parental lipid) or phosphatidylserine (PS, negatively charged like SGG) liposomes did not. All of these results suggested the role of human sperm SGG in ZP binding. Copyright 2001 Wiley-Liss, Inc.

Probing the carbonyl functionality of a petroleum resin and asphaltene through oximation and schiff base formation in conjunction with N-15 NMR

USGS Publications Warehouse

Thorn, Kevin A.; Cox, Larry G.

2015-01-01

Despite recent advances in spectroscopic techniques, there is uncertainty regarding the nature of the carbonyl groups in the asphaltene and resin fractions of crude oil, information necessary for an understanding of the physical properties and environmental fate of these materials. Carbonyl and hydroxyl group functionalities are not observed in natural abundance 13C nuclear magnetic resonance (NMR) spectra of asphaltenes and resins and therefore require spin labeling techniques for detection. In this study, the carbonyl functionalities of the resin and asphaltene fractions from a light aliphatic crude oil that is the source of groundwater contamination at the long term USGS study site near Bemidji, Minnesota, have been examined through reaction with 15N-labeled hydroxylamine and aniline in conjunction with analysis by solid and liquid state 15N NMR. Ketone groups were revealed through 15N NMR detection of their oxime and Schiff base derivatives, and esters through their hydroxamic acid derivatives. Anilinohydroquinone adducts provided evidence for quinones. Some possible configurations of the ketone groups in the resin and asphaltene fractions can be inferred from a consideration of the likely reactions that lead to heterocyclic condensation products with aniline and to the Beckmann reaction products from the initially formed oximes. These include aromatic ketones and ketones adjacent to quaternary carbon centers, β-hydroxyketones, β-diketones, and β-ketoesters. In a solid state cross polarization/magic angle spinning (CP/MAS) 15N NMR spectrum recorded on the underivatized asphaltene as a control, carbazole and pyrrole-like nitrogens were the major naturally abundant nitrogens detected.

Isotopic measurements (C,N,O) of detonation soot produced from labeled and unlabeled Composition B-3 indicate source of solid carbon residues

NASA Astrophysics Data System (ADS)

Podlesak, David; Manner, Virginia; Amato, Ronald; Dattelbaum, Dana; Gusavsen, Richard; Huber, Rachel

2017-06-01

Detonation of HE is an exothermic process whereby metastable complex molecules are converted to simple stable molecules such as H2 O, N2, CO, CO2, and solid carbon. The solid carbon contains various allotropes such as detonation nanodiamonds, graphite, and amorphous carbon. It is well known that certain HE formulations such as Composition B (60% RDX, 40% TNT) produce greater amounts of solid carbon than other more oxygen-balanced formulations. To develop a greater understanding of how formulation and environment influence solid carbon formation, we synthesized TNT and RDX with 13 C and 15 N at levels slightly above natural abundance levels. Synthesized RDX and TNT were mixed at a ratio of 60:40 to form Composition B and solid carbon residues were collected from detonations of isotopically-labeled as well as un-labelled Composition B. The raw HE and detonation residues were analyzed isotopically for C, N, O isotopic compositions. We will discuss differences between treatments groups as a function of formulation and environment. LA-UR - 17-21266.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

PubMed

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

2016-01-22

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Biodistribution and radiation dosimetry of LMI1195: first-in-human study of a novel 18F-labeled tracer for imaging myocardial innervation.

PubMed

Sinusas, Albert J; Lazewatsky, Joel; Brunetti, Jacqueline; Heller, Gary; Srivastava, Ajay; Liu, Yi-Hwa; Sparks, Richard; Puretskiy, Andrey; Lin, Shu-fei; Crane, Paul; Carson, Richard E; Lee, L Veronica

2014-09-01

A novel (18)F-labeled ligand for the norepinephrine transporter (N-[3-bromo-4-(3-(18)F-fluoro-propoxy)-benzyl]-guanidine [LMI1195]) is in clinical development for mapping cardiac nerve terminals in vivo using PET. Human safety, whole-organ biodistribution, and radiation dosimetry of LMI1195 were evaluated in a phase 1 clinical trial. Twelve healthy subjects at 3 clinical sites were injected intravenously with 150-250 MBq of LMI1195. Dynamic PET images were obtained over the heart for 10 min, followed by sequential whole-body images for approximately 5 h. Blood samples were obtained, and heart rate, electrocardiogram, and blood pressure were monitored before and during imaging. Residence times were determined from multiexponential regression of organ region-of-interest data normalized by administered activity (AA). Radiation dose estimates were calculated using OLINDA/EXM. Myocardial, lung, liver, and blood-pool standardized uptake values were determined at different time intervals. No adverse events due to LMI1195 were seen. Blood radioactivity cleared quickly, whereas myocardial uptake remained stable and uniform throughout the heart over 4 h. Liver and lung activity cleared relatively rapidly, providing favorable target-to-background ratios for cardiac imaging. The urinary bladder demonstrated the largest peak uptake (18.3% AA), followed by the liver (15.5% AA). The mean effective dose was 0.026 ± 0.0012 mSv/MBq. Approximately 1.6% AA was seen in the myocardium initially, remaining above 1.5% AA (decay-corrected) through 4 h after injection. The myocardium-to-liver ratio was approximately unity initially, increasing to more than 2 at 4 h. These preliminary data suggest that LMI1195 is well tolerated and yields a radiation dose comparable to that of other commonly used PET radiopharmaceuticals. The kinetics of myocardial and adjacent organ activity suggest that cardiac imaging should be possible with acceptable patient radiation dose. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Mineralization of RDX-derived nitrogen to N2 via denitrification in coastal marine sediments.

PubMed

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

2015-02-17

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common constituent of military explosives. Despite RDX contamination at numerous U.S. military facilities and its mobility to aquatic systems, the fate of RDX in marine systems remains largely unknown. Here, we provide RDX mineralization pathways and rates in seawater and sediments, highlighting for the first time the importance of the denitrification pathway in determining the fate of RDX-derived N. (15)N nitro group labeled RDX ((15)N-[RDX], 50 atom %) was spiked into a mesocosm simulating shallow marine conditions of coastal Long Island Sound, and the (15)N enrichment of N2 (δ(15)N2) was monitored via gas bench isotope ratio mass spectrometry (GB-IRMS) for 21 days. The (15)N tracer data were used to model RDX mineralization within the context of the broader coastal marine N cycle using a multicompartment time-stepping model. Estimates of RDX mineralization rates based on the production and gas transfer of (15)N2O and (15)N2 ranged from 0.8 to 10.3 μmol d(-1). After 22 days, 11% of the added RDX had undergone mineralization, and 29% of the total removed RDX-N was identified as N2. These results demonstrate the important consideration of sediment microbial communities in management strategies addressing cleanup of contaminated coastal sites by military explosives.

Evidence for metabolic activity of airborne bacteria

NASA Technical Reports Server (NTRS)

Dimmick, R. L.; Wolochow, H.; Chatigny, M. A.; Straat, P. A.; Schrot, J. R.; Levin, G. V.

1974-01-01

Aerosols of the bacterium Serratia marcescens, and of uniformly labelled C-14 glucose, were created simultaneously and mixed in tubing leading to an aerosol chamber. During a subsequent period of about 5 hrs, C-14O2 was produced unequivocally within the chamber, and insoluble, labelled material within the suspended particles first increased, then decreased.

Carbon and nitrogen partitioning during the post-anthesis period is conditioned by N fertilisation and sink strength in three cereals.

PubMed

Aranjuelo, I; Cabrera-Bosquet, L; Araus, J L; Nogués, S

2013-01-01

Further knowledge of the processes conditioning nitrogen use efficiency (NUE) is of great relevance to crop productivity. The aim of this paper was characterise C and N partitioning during grain filling and their implications for NUE. Cereals such as bread wheat (Triticum aestivum L. cv Califa sur), triticale (× Triticosecale Wittmack cv. Imperioso) and tritordeum (× Tritordeum Asch. & Graebn line HT 621) were grown under low (LN, 5 mm NH(4) NO(3)) and high (HN, 15 mm NH(4)NO(3)) N conditions. We conducted simultaneous double labelling ((12)CO(2) and (15)NH(4) (15)NO(3)) in order to characterise C and N partitioning during grain filling. Although triticale plants showed the largest total and ear dry matter values in HN conditions, the large investment in shoot and root biomass negatively affected ear NUE. Tritordeum was the only genotype that increased NUE in both N treatments (NUE(total)), whereas in wheat, no significant effect was detected. N labelling revealed that N fertilisation during post-anthesis was more relevant for wheat and tritordeum grain filling than for triticale. The study also revealed that the investments of C and N in flag leaves and shoots, together with the 'waste' of photoassimilates in respiration, conditioned the NUE of plants, and especially under LN. These results suggest that C and N use by these plants needs to be improved in order to increase ear C and N sinks, especially under LN. It is also remarkable that even though tritordeum shows the largest increase in NUE, the low yield of this cereal limits its agronomic value. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

Quantification of multiple simultaneously occurring nitrogen flows in the euphotic ocean

NASA Astrophysics Data System (ADS)

Xu, Min Nina; Wu, Yanhua; Zheng, Li Wei; Zheng, Zhenzhen; Zhao, Huade; Laws, Edward A.; Kao, Shuh-Ji

2017-03-01

The general features of the N cycle in the sunlit region of the ocean are well known, but methodological difficulties have previously confounded simultaneous quantification of transformation rates among the many different forms of N, e.g., ammonium (NH4+), nitrite (NO2-), nitrate (NO3-), and particulate/dissolved organic nitrogen (PN/DON). However, recent advances in analytical methodology have made it possible to employ a convenient isotope labeling technique to quantify in situ fluxes among oft-measured nitrogen species within the euphotic zone. Addition of a single 15N-labeled NH4+ tracer and monitoring of the changes in the concentrations and isotopic compositions of the total dissolved nitrogen (TDN), PN, NH4+, NO2-, and NO3- pools allowed us to quantify the 15N and 14N fluxes simultaneously. Constraints expressing the balance of 15N and 14N fluxes between the different N pools were expressed in the form of simultaneous equations, the unique solution of which via matrix inversion yielded the relevant N fluxes, including rates of NH4+, NO2-, and NO3- uptake; ammonia oxidation; nitrite oxidation; DON release; and NH4+ uptake by bacteria. The matrix inversion methodology that we used was designed specifically to analyze the results of incubations under simulated in situ conditions in the euphotic zone. By taking into consideration simultaneous fluxes among multiple N pools, we minimized potential artifacts caused by non-targeted processes in traditional source-product methods. The proposed isotope matrix method facilitates post hoc analysis of data from on-deck incubation experiments and can be used to probe effects of environmental factors (e.g., pH, temperature, and light) on multiple processes under controlled conditions.

Ecophysiological adjustment of two Sphagnum species in response to anthropogenic nitrogen deposition.

PubMed

Wiedermann, Magdalena M; Gunnarsson, Urban; Ericson, Lars; Nordin, Annika

2009-01-01

Here, it was investigated whether Sphagnum species have adjusted their nitrogen (N) uptake in response to the anthropogenic N deposition that has drastically altered N-limited ecosystems, including peatlands, worldwide. A lawn species, Sphagnum balticum, and a hummock species, Sphagnum fuscum, were collected from three peatlands along a gradient of N deposition (2, 8 and 12 kg N ha(-1) yr(-1)). The mosses were subjected to solutions containing a mixture of four N forms. In each solution one of these N forms was labeled with (15)N (namely (15)NH(+)(4), (15)NO(-)(3) and the amino acids [(15)N]alanine (Ala) and [(15)N]glutamic acid (Glu)). It was found that for both species most of the N taken up was from , followed by Ala, Glu, and very small amounts from NO(-)(3). At the highest N deposition site N uptake was reduced, but this did not prevent N accumulation as free amino acids in the Sphagnum tissues. The reduced N uptake may have been genetically selected for under the relatively short period with elevated N exposure from anthropogenic sources, or may have been the result of plasticity in the Sphagnum physiological response. The negligible Sphagnum NO(-)(3) uptake may make any NO(-)(3) deposited readily available to co-occurring vascular plants.

Rapid monitoring of intermediate states and mass balance of nitrogen during denitrification by means of cavity enhanced Raman multi-gas sensing.

PubMed

Keiner, Robert; Herrmann, Martina; Küsel, Kirsten; Popp, Jürgen; Frosch, Torsten

2015-03-15

The comprehensive investigation of changes in N cycling has been challenging so far due to difficulties with measuring gases such as N2 and N2O simultaneously. In this study we introduce cavity enhanced Raman gas spectroscopy as a new analytical methodology for tracing the stepwise reduction of (15)N-labelled nitrate by the denitrifying bacteria Pseudomonas stutzeri. The unique capabilities of Raman multi-gas analysis enabled real-time, continuous, and non-consumptive quantification of the relevant gases ((14)N2, (14)N2O, O2, and CO2) and to trace the fate of (15)N-labeled nitrate substrate ((15)N2, (15)N2O) added to a P. stutzeri culture with one single measurement. Using this new methodology, we could quantify the kinetics of the formation and degradation for all gaseous compounds (educts and products) and thus study the reaction orders. The gas quantification was complemented with the analysis of nitrate and nitrite concentrations for the online monitoring of the total nitrogen element budget. The simultaneous quantification of all gases also enabled the contactless and sterile online acquisition of the pH changes in the P. stutzeri culture by the stoichiometry of the redox reactions during denitrification and the CO2-bicarbonate equilibrium. Continuous pH monitoring - without the need to insert an electrode into solution - elucidated e.g. an increase in the slope of the pH value coinciding with an accumulation of nitrite, which in turn led to a temporary accumulation of N2O, due to an inhibition of nitrous oxide reductase. Cavity enhanced Raman gas spectroscopy has a high potential for the assessment of denitrification processes and can contribute substantially to our understanding of nitrogen cycling in both natural and agricultural systems. Copyright © 2015 Elsevier B.V. All rights reserved.

21 CFR 801.15 - Medical devices; prominence of required label statements.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical devices; prominence of required label statements. 801.15 Section 801.15 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES LABELING General Labeling Provisions § 801.15 Medical devices; prominence of required label statements. (a...

21 CFR 201.15 - Drugs; prominence of required label statements.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Drugs; prominence of required label statements. 201.15 Section 201.15 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL LABELING General Labeling Provisions § 201.15 Drugs; prominence of required label statements. (a) A word, statement,...

Refractive improvements and safety with topography-guided corneal crosslinking for keratoconus: 1-year results.

PubMed

Nordström, Maria; Schiller, Maria; Fredriksson, Anneli; Behndig, Anders

2017-07-01

To assess the refractive improvements and the corneal endothelial safety of an individualised topography-guided regimen for corneal crosslinking in progressive keratoconus. An open-label prospective randomised clinical trial was performed at the Department of Clinical Sciences, Ophthalmology, Umeå University Hospital, Umeå, Sweden. Thirty-seven patients (50 eyes) with progressive keratoconus planned for corneal crosslinking were included. The patients were randomised to topography-guided crosslinking (photorefractive intrastromal crosslinking (PiXL); n=25) or uniform 9 mm crosslinking (corneal collagen crosslinking (CXL); n=25). Visual acuity, refraction, keratometry (K1, K2 and K max ) and corneal endothelial morphometry were assessed preoperatively and at 1, 3, 6 and 12 months postoperatively. The PiXL treatment involved an asymmetrical treatment zone centred on the area of maximum corneal steepness with treatment energies ranging from 7.2 to 15.0 J/cm 2 ; the CXL treatment was a uniform 9 mm 5.4 J/cm 2 pulsed crosslinking. The main outcome measures were changes in refractive errors and corneal endothelial cell density. The spherical refractive errors decreased (p<0.05) and the visual acuity improved (p<0.01) at 3, 6 and 12 months after PiXL, but not after CXL. The between-groups differences, however, were not significant. K2 and K max decreased at 3, 6 and 12 months after PiXL (p<0.01), but not after CXL (p<0.01 when comparing the two treatments). No corneal endothelial cell loss was seen after either treatment. Individualised topography-based crosslinking treatment centred on the ectatic cone has the potential to improve the corneal shape in keratoconus with decreased spherical refractive errors and improved visual acuity, without damage to the corneal endothelium. NCT02514200, Results. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Functional Single-Cell Approach to Probing Nitrogen-Fixing Bacteria in Soil Communities by Resonance Raman Spectroscopy with 15N2 Labeling.

PubMed

Cui, Li; Yang, Kai; Li, Hong-Zhe; Zhang, Han; Su, Jian-Qiang; Paraskevaidi, Maria; Martin, Francis L; Ren, Bin; Zhu, Yong-Guan

2018-04-17

Nitrogen (N) fixation is the conversion of inert nitrogen gas (N 2 ) to bioavailable N essential for all forms of life. N 2 -fixing microorganisms (diazotrophs), which play a key role in global N cycling, remain largely obscure because a large majority are uncultured. Direct probing of active diazotrophs in the environment is still a major challenge. Herein, a novel culture-independent single-cell approach combining resonance Raman (RR) spectroscopy with 15 N 2 stable isotope probing (SIP) was developed to discern N 2 -fixing bacteria in a complex soil community. Strong RR signals of cytochrome c (Cyt c, frequently present in diverse N 2 -fixing bacteria), along with a marked 15 N 2 -induced Cyt c band shift, generated a highly distinguishable biomarker for N 2 fixation. 15 N 2 -induced shift was consistent well with 15 N abundance in cell determined by isotope ratio mass spectroscopy. By applying this biomarker and Raman imaging, N 2 -fixing bacteria in both artificial and complex soil communities were discerned and imaged at the single-cell level. The linear band shift of Cyt c versus 15 N 2 percentage allowed quantification of N 2 fixation extent of diverse soil bacteria. This single-cell approach will advance the exploration of hitherto uncultured diazotrophs in diverse ecosystems.

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

PubMed Central

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

2016-01-01

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

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

USGS Publications Warehouse

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

2013-01-01

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

A universal sample manipulator with 50 kV negative bias

NASA Astrophysics Data System (ADS)

Kenny, M. J.; Wielunski, L. S.; Scott, M. D.; Clissold, R. A.; Stevenson, D.; Baxter, G.

1991-04-01

A manipulator incorporating a number of novel features has been built for a research ion implanter. The system is designed to enable uniform dose implantation of both planar and nonplanar surfaces by incorporating one translational and two rotational degrees of freedom. Negative target bias of up to 50 kV may be applied to the target, thus increasing the ion energy by this amount. The target chamber and all external manipulator controls are grounded. With the exception of the high voltage power supply, cable and feedthrough, all high voltage components are within the vacuum system. A secondary electron suppression cage which can be held at a negative bias of up to 60 kV relative to the chamber (i.e. 10 kV relative to the manipulator) surrounds the manipulator. Performance has been evaluated using 15N ions and nuclear reaction analysis through 15N(p,α) 12C to profile ion concentrations for dose uniformity and for ion depth at elevated target potentials.

Use of DNA barcoding to reveal species composition of convenience seafood.

PubMed

Huxley-Jones, Elizabeth; Shaw, Jennifer L A; Fletcher, Carly; Parnell, Juliette; Watts, Phillip C

2012-04-01

Increased education of consumers can be an effective tool for conservation of commercially harvested marine species when product labeling is accurate and allows an informed choice. However, generic labeling (e.g., as white fish or surimi) and mislabeling of seafood prevents this and may erode consumer confidence in seafood product labels in general. We used DNA barcoding to identify the species composition of two types of convenience seafood (i.e., products processed for ease of consumption): fish fingers (long pieces of fish covered with bread crumbs or batter, n = 241) and seafood sticks (long pieces of cooked fish, n = 30). In products labeled as either white fish or surimi, four teleost species were present. Less than 1.5% of fish fingers with species-specific information were mislabeled. Results of other studies show substantially more mislabeling (e.g., >25%) of teleost products, which likely reflects the lower economic gains associated with mislabeling of convenience seafood compared with whole fillets. In addition to species identification, seafood product labels should be required to contain information about, for example, harvesting practices, and our data indicate that consumers can have reasonable confidence in the accuracy of the labels of convenience seafood and thus select brands on the basis of information about current fisheries practice. ©2012 Society for Conservation Biology.

Visualizing carbon and nitrogen transfer in the tripartite symbiosis of Fagus sylvatica, ectomycorrhizal fungi and soil microorganisms using NanoSIMS

NASA Astrophysics Data System (ADS)

Mayerhofer, Werner; Dietrich, Marlies; Schintlmeister, Arno; Gabriel, Raphael; Gorka, Stefan; Wiesenbauer, Julia; Martin, Victoria; Schweiger, Peter; Reipert, Siegfried; Weidinger, Marieluise; Richter, Andreas; Woebken, Dagmar; Kaiser, Christina

2016-04-01

Translocation of recently photoassimilated plant carbon (C) into soil via root exudates or mycorrhizal fungi is key to understand global carbon cycling. Plants support symbiotic fungi and soil microorganisms with recent photosynthates to get access to essential elements, such as nitrogen (N) and phosphorus. While a 'reciprocal reward strategy' (plants trade C in exchange for nutrients from the fungus) has been shown for certain types of mycorrhizal associations, only little is known about the mechanisms of C and N exchange between mycorrhizal fungal hyphae and soil bacteria. Our understanding of the underlying mechanisms is hampered by the fact that C and N transfer between plants, mycorrhizal fungi and soil bacteria takes place at the micrometer scale, which makes it difficult to explore at the macro scale. In this project we intended to analyse carbon and nitrogen flows between roots of beech trees (Fagus sylvatica), their associated ectomycorrhizal fungi and bacterial community. In order to visualize this nutrient flow at a single cell level, we used a stable isotope double labelling (13C and 15N) approach. Young mycorrhizal beech trees were transferred from a forest to split-root boxes, consisting of two compartments separated by a membrane (35 μm mesh size) which was penetrable for hyphae but not for plant roots. After trees and mycorrhizal fungi were allowed to grow for one year in these boxes, 15N-labelled nitrogen solution was added only to the root-free compartment to allow labelled nitrogen supply only through the fungal network. 13C- labelled carbon was applied by exposing the plants to a 13CO2 gas atmosphere for 8 hours. Spatial distribution of the isotopic label was visualised at the microscale in cross sections of mycorrhizal root-tips (the plant/mycorrhizal fungi interface) and within and on the surface of external mycorrhizal hyphae (the fungi/soil bacteria interface) using nanoscale secondary ion mass spectrometry (NanoSIMS). Corresponding morphological structures were established using light microscopy and scanning electron microscopy. In addition, isotopic signals in plant tissue as well as in fungal and soil microbial communities were traced by EA-IRMS and GC-C-IRMS of 13C phospholipid fatty acid, respectively. Our NanoSIMS images demonstrate a rapid transfer of photoassimilated plant C from the root's central cylinder to 1) ectomycorrhizal fungal cells in the Hartig net in the root cortex, and 2) to external ectomycorrhizal hyphae residing in the root-free compartment. In the cross-section of the mycorrhizal root, 13C enrichment was spatially correlated to 15N enrichment indicating a strongly controlled exchange of C and N between plant and fungus. Overall, our study shows the potential of NanoSIMS imaging as a tool for getting insight into mechanisms of plant-soil interactions by visualizing in situ C and N flows between plants, fungi and soil microbes at the microscale.

Enhanced N-Transfer from a Soybean to Maize by Vesicular Arbuscular Mycorrhizal (VAM) Fungi.

PubMed

van Kessel, C; Singleton, P W; Hoben, H J

1985-10-01

Using a split-root technique, roots of soybean plants were divided between two pots. In one of the two pots, two maize plants were grown and half of those pots were inoculated with the vesicular arbuscular mycorrhizal (VAM) fungus, Glomus fasciculatus. Fifty-two days after planting, (15)N-labeled ammonium sulfate was applied to the pots which contained only soybean roots. Forty-eight hours after application, significantly higher values for atom per cent (15)N excess were found in roots and leaves of VAM-infected maize plants as compared with the non-VAM-infected maize plants. Results indicated that VAM fungi did enhance N transfer from one plant to another.

Effects of interpretive nutrition labels on consumer food purchases: the Starlight randomized controlled trial.

PubMed

Ni Mhurchu, Cliona; Volkova, Ekaterina; Jiang, Yannan; Eyles, Helen; Michie, Jo; Neal, Bruce; Blakely, Tony; Swinburn, Boyd; Rayner, Mike

2017-03-01

Background: Nutrition labeling is a prominent policy to promote healthy eating. Objective: We aimed to evaluate the effects of 2 interpretive nutrition labels compared with a noninterpretive label on consumer food purchases. Design: In this parallel-group randomized controlled trial, we enrolled household shoppers across New Zealand who owned smartphones and were aged ≥18 y. Eligible participants were randomly assigned (1:1:1) to receive either traffic light labels (TLLs), Health Star Rating labels (HSRs), or a control [nutrition information panel (NIP)]. Smartphone technology allowed participants to scan barcodes of packaged foods and to receive allocated labels on their smartphone screens. The primary outcome was the mean healthiness of all packaged food purchases over the 4-wk intervention period, which was measured by using the Food Standards Australia New Zealand Nutrient Profiling Scoring Criterion (NPSC). Results: Between October 2014 and November 2015, 1357 eligible shoppers were randomly assigned to TLL ( n = 459), HSR ( n = 443), or NIP ( n = 455) labels. Overall difference in the mean transformed NPSC score for the TLL group compared with the NIP group was -0.20 (95% CI: -0.94, 0.54; P = 0.60). The corresponding difference for HSR compared with NIP was -0.60 (95% CI: -1.35, 0.15; P = 0.12). In an exploratory per-protocol analysis of participants who used the labeling intervention more often than average ( n = 423, 31%), those who were assigned to TLL and HSR had significantly better NPSC scores [TLL compared with NIP: -1.33 (95% CI: -2.63, -0.04; P = 0.04); HSR compared with NIP: -1.70 (95% CI: -2.97, -0.43; P = 0.01)]. Shoppers who were randomly assigned to HSR and TLL also found the labels significantly more useful and easy to understand than the NIP (all P values <0.001). Conclusions: At the relatively low level of use observed in this trial, interpretive nutrition labels had no significant effect on food purchases. However, shoppers who used interpretive labels found them to be significantly more useful and easy to understand, and compared with frequent NIP users, frequent TLL and HSR users had significantly healthier food purchases. This trial was registered at the Australian New Zealand Clinical Trials Registry (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=366446&isReview=true) as ACTRN12614000644662. © 2017 American Society for Nutrition.

Inoculation of Bacillus sphaericus UPMB-10 to Young Oil Palm and Measurement of Its Uptake of Fixed Nitrogen Using the 15N Isotope Dilution Technique

PubMed Central

Zakry, Fitri Abdul Aziz; Shamsuddin, Zulkifli H.; Rahim, Khairuddin Abdul; Zakaria, Zin Zawawi; Rahim, Anuar Abdul

2012-01-01

There are increasing applications of diazotrophic rhizobacteria in the sustainable agriculture system. A field experiment on young immature oil palm was conducted to quantify the uptake of N derived from N2 fixation by the diazotroph Bacillus sphaericus strain UPMB-10, using the 15N isotope dilution method. Eight months after 15N application, young immature oil palms that received 67% of standard N fertilizer application together with B. sphaericus inoculation had significantly lower 15N enrichment than uninoculated palms that received similar N fertilizers. The dilution of labeled N served as a marker for the occurrence of biological N2 fixation. The proportion of N uptake that was derived from the atmosphere was estimated as 63% on the whole plant basis. The inoculation process increased the N and dry matter yields of the palm leaflets and rachis significantly. Field planting of young, immature oil palm in soil inoculated with B. sphaericus UPMB-10 might mitigate inorganic fertilizer-N application through supplementation by biological nitrogen fixation. This could be a new and important source of nitrogen biofertilizer in the early phase of oil palm cultivation in the field. PMID:22446306

Mineral nitrogen transformations in and under seasonal snow in a high-elevation catchment in the Rocky Mountains, United States

USGS Publications Warehouse

Williams, Mark W.; Brooks, Paul D.; Mosier, Arvin; Tonnessen, Kathy A.

1996-01-01

In an effort to understand sources of nitrate (NO3−) in surface waters of high-elevation catchments, nitrogen (N) transformations in and under seasonal snow were investigated from 1993 to 1995 on Niwot Ridge, an alpine ecosystem at 3,500 m located in the Colorado Front Range of the Rocky Mountains. Ammonium (NH4+) and NO3− labeled with 15N applied as nonconservative tracers to the snow showed no evidence of nitrification in the snowpack. Furthermore, NH4+ movement through the amended snowpack was highly correlated with a conservative chloride tracer (r2 = 0.99). In an unamended snowpack NH4+ concentrations in meltwater before contact with the ground were highly correlated with NO3− concentrations (r2 = 0.98), which is consistent with no nitrification in the snowpack. The isotopically labeled 15NH+4applied to the snowpack was found in underlying soils, showing that NH4+ released from snow can be rapidly immobilized. Resin bag (mixed-bed ion-exchange resins) measurements (n = 22) showed that 80% of the mobile inorganic N in unamended subnivial soils was NO3−. Measurements of KCl-extractable inorganic N from surface soils showed that highest values were prior to the initiation of snowmelt and lowest values were during the growing season. The natural δ15N abundance of unamended soils was negative and ranged from −12 to −2, suggesting that atmospheric deposition of δ15N-depleted N is an important component of N cycling in these alpine soils. These results suggest that soil mineralization under seasonal snow, rather than snowmelt release of NO3−, may control NO3− concentrations in surface waters of high-elevation catchments.

The influence of labeling the vegetable content of snack food on children's taste preferences: a pilot study.

PubMed

Pope, Lizzy; Wolf, Randi L

2012-01-01

This pilot study examined whether informing children of the presence of vegetables in select snack food items alters taste preference. A random sample of 68 elementary and middle school children tasted identical pairs of 3 snack food items containing vegetables. In each pair, 1 sample's label included the food's vegetable (eg, broccoli gingerbread spice cake), and 1 sample's label did not (eg, gingerbread spice cake). Participants reported whether the samples tasted the same, or whether they preferred one sample. Frequency of vegetable consumption was also assessed. Taste preferences did not differ for the labeled versus the unlabeled sample of zucchini chocolate chip bread, χ(2) (2, n = 68) = 3.21, P = .20 or broccoli gingerbread spice cake χ(2) (2, n = 68) = 2.15, P = .34. However, students preferred the unlabeled cookies (ie, chocolate chip cookies) over the vegetable-labeled version (ie, chickpea chocolate chip cookies), χ(2) = (2, n = 68) 9.21, P = .01. Chickpeas were consumed less frequently (81% had not tried in past year) as compared to zucchini and broccoli. Informing children of the presence of vegetables hidden within snack food may or may not alter taste preference and may depend on the frequency of prior exposure to the vegetable. Copyright © 2012 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

Novel isotopic N, N-Dimethyl Leucine (iDiLeu) Reagents Enable Absolute Quantification of Peptides and Proteins Using a Standard Curve Approach

NASA Astrophysics Data System (ADS)

Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

2015-01-01

Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N, N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

Understanding N timing in corn yield and fertilizer N recovery: An insight from an isotopic labeled-N determination

PubMed Central

de Almeida, Rodrigo Estevam Munhoz; Pierozan Junior, Clovis; Lago, Bruno Cocco; Trivelin, Paulo Cesar Ocheuze

2018-01-01

Early fertilizer nitrogen (N) application on cover crops or their residues during the off-season is a practice adopted in Brazil subtropical conditions under no-tillage corn (Zea mays L.) systems. However, the effect of early N application on yield, plant N content, and N recovery efficiency (NRE) for corn is not yet well documented. Five fertilizer N timings in an oat-corn system were evaluated in two studies utilizing an isotopic-labeled N determination, 15N isotope. The N fertilization timings were: (i) oat tillering, (ii) 15 days before corn planting time, over the oat residues, (iii) at corn planting time, (iv) in-season at the three-leaf growth stage (V3), and (v) in-season split application at V3 and six-leaf (V6) growth stages. Based on the statistical analysis, the N fertilization timings were separated into three groups: 1) N-OATS, designated to N applied at oat; 2) N-PLANT, referred to pre-plant and planting N applications; and 3) N-CORN, designated to in-season corn N applications. Corn yield was not affected by the N fertilization timing. However, the N-CORN N fertilization timings enhanced NRE by 17% and 35% and final N recovery system (plant plus soil) by 16% and 24% all relative to N-OATS and N-PLANT groups, respectively. Overall, N-OATS resulted in the largest N derived from fertilizer (NDFF) amount in the deeper soil layer, in overall a delta of 10 kg N ha-1 relative to the rest of the groups. Notwithstanding corn yield was not affected, early N fertilization under subtropical conditions is not a viable option since NRE was diminished and the non-recovery N increased relative to the in-season N applications. PMID:29462178

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

PubMed

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

2007-10-01

Utilization of long-range (1)H--(15)N heteronuclear chemical shift correlation has continually grown in importance since the first applications were reported in 1995. More recently, indirect covariance NMR methods have been introduced followed by the development of unsymmetrical indirect covariance processing methods. The latter technique has been shown to allow the calculation of hyphenated 2D NMR data matrices from more readily acquired nonhyphenated 2D NMR spectra. We recently reported the use of unsymmetrical indirect covariance processing to combine (1)H--(13)C GHSQC and (1)H--(15)N GHMBC long-range spectra to yield a (13)C--(15)N HSQC-HMBC chemical shift correlation spectrum that could not be acquired in a reasonable period of time without resorting to (15)N-labeled molecules. We now report the unsymmetrical indirect covariance processing of (1)H--(13)C GHMBC and (1)H--(15)N IMPEACH spectra to afford a (13)C--(15)N HMBC-IMPEACH spectrum that has the potential to span as many as six to eight bonds. Correlations for carbon resonances long-range coupled to a protonated carbon in the (1)H--(13)C HMBC spectrum are transferred via the long-range (1)H--(15)N coupling pathway in the (1)H--(15)N IMPEACH spectrum to afford a much broader range of correlation possibilities in the (13)C--(15)N HMBC-IMPEACH correlation spectrum. The indole alkaloid vincamine is used as a model compound to illustrate the application of the method. (c) 2007 John Wiley & Sons, Ltd.

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

USDA-ARS?s Scientific Manuscript database

The absorption and plasma elimination of vitamin K was investigated by uniformly labeling phylloquinone in kale with carbon-13 and feeding the kale to study subjects. Seven healthy volunteers ingested a single 400 g serving of kale with 30 g vegetable oil. The kale provided 156 nmol of phylloquino...

A robust heteronuclear dipolar recoupling method comparable to TEDOR for proteins in magic-angle spinning solid-state NMR

NASA Astrophysics Data System (ADS)

Zhang, Zhengfeng; Li, Jianping; Chen, Yanke; Xie, Huayong; Yang, Jun

2017-12-01

In this letter, we propose a robust heteronuclear dipolar recoupling method for proteins in magic-angle spinning (MAS) solid-state NMR. This method is as simple, robust and efficient as the well-known TEDOR in the aspect of magnetization transfer between 15N and 13C. Deriving from our recent band-selective dual back-to-back pulses (DBP) (Zhang et al., 2016), this method uses new phase-cycling schemes to realize broadband DBP (Bro-DBP). For broadband 15N-13C magnetization transfer (simultaneous 15N → 13C‧ and 15N → 13Cα), Bro-DBP has almost the same 15N → 13Cα efficiency while offers 30-40% enhancement on 15N → 13C‧ transfer, compared to TEDOR. Besides, Bro-DBP can also be used as a carbonyl (13C‧)-selected method, whose 15N → 13C‧ efficiency is up to 1.7 times that of TEDOR and is also higher than that of band-selective DBP. The performance of Bro-DBP is demonstrated on the N-formyl-[U-13C,15N]-Met-Leu-Phe-OH (fMLF) peptide and the U-13C, 15N labeled β1 immunoglobulin binding domain of protein G (GB1) microcrystalline protein. Since Bro-DBP is as robust, simple and efficient as TEDOR, we believe it is very useful for protein studies in MAS solid-state NMR.

49 CFR 172.411 - EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels..., 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label. (a) Except for size and color....5 and EXPLOSIVE 1.6 labels must be as follows: EXPLOSIVE 1.4: EC02MR91.016 EXPLOSIVE 1.5: EC02MR91...

Macrofauna regulate heterotrophic bacterial carbon and nitrogen incorporation in low-oxygen sediments

PubMed Central

Hunter, William R; Veuger, Bart; Witte, Ursula

2012-01-01

Oxygen minimum zones (OMZs) currently impinge upon >1 million km2 of sea floor and are predicted to expand with climate change. We investigated how changes in oxygen availability, macrofaunal biomass and retention of labile organic matter (OM) regulate heterotrophic bacterial C and N incorporation in the sediments of the OMZ-impacted Indian continental margin (540–1100 m; [O2]=0.35–15 μmol l−1). In situ pulse-chase experiments traced 13C:15N-labelled phytodetritus into bulk sediment OM and hydrolysable amino acids, including the bacterial biomarker 𝒟-alanine. Where oxygen availability was lowest ([O2]=0.35 μmol l−1), metazoan macrofauna were absent and bacteria assimilated 30–90% of the labelled phytodetritus within the sediment. At higher oxygen levels ([O2]=2–15 μmol l−1) the macrofaunal presence and lower phytodetritus retention with the sediment occur concomitantly, and bacterial phytodetrital incorporation was reduced and retarded. Bacterial C and N incorporation exhibited a significant negative relationship with macrofaunal biomass across the OMZ. We hypothesise that fauna–bacterial interactions significantly influence OM recycling in low-oxygen sediments and need to be considered when assessing the consequences of global change on biogeochemical cycles. PMID:22592818

Development of Antibodies Against Novel Cell Surface Proteins In Hormone Refractory Prostate Cancer. Addendum

DTIC Science & Technology

2011-07-01

marker of hormone refractory metastatic prostate cancer. Clin Cancer Res, 2005. 15: 2237- 43 . 3. Tomita K, van Bokhoven A, van Leenders GJ, Ruijter...Santa Cruz Biotechnology), vimentin, Ki-67 (DakoCytomation) and caspase-3 (Cell Signaling Technology). Flow cytometry was performed with N...transduced cells were labeled with N-cadherin–specific antibody and sorted by flow cytometry , gating for a GFP-positive, N-cadherinlow population. The

Structural investigation of Titan tholins by solution-state 1H, 13C, and 15N NMR: one-dimensional and decoupling experiments.

PubMed

He, Chao; Lin, Guangxin; Upton, Kathleen T; Imanaka, Hiroshi; Smith, Mark A

2012-05-17

Titan, the largest moon of Saturn, is enveloped in a reddish brown organic haze. Titan haze is presumed to be formed from methane and nitrogen (CH(4) and N(2)) in Titan's upper atmosphere through energetic photochemistry and particle bombardment. Though Titan haze has been directly investigated using methods including the Cassini mission, its formation mechanism and the contributing chemical structures and prebiotic potential are still not well developed. We report here the structural investigation of the (13)C and (15)N labeled, simulated Titan haze aerosol (tholin) by solution-state NMR. The one-dimensional (1)H, (13)C, and (15)N NMR spectra and decoupling experiments indicate that the tholin sample contains amine, nitrile, imine, and N-heteroaromatic compounds of tremendous import in understanding complex organic chemistry in anaerobic, extraterrestrial environments.

Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA.

PubMed

Liu, Yu; Holmstrom, Erik; Yu, Ping; Tan, Kemin; Zuo, Xiaobing; Nesbitt, David J; Sousa, Rui; Stagno, Jason R; Wang, Yun-Xing

2018-05-01

Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13 C 15 N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.

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

PubMed

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

2014-01-21

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

Quantification of transformation rates of soil amino sugars and amino acids by a novel isotope pool dilution approach via liquid chromatography/high resolution mass spectrometry (LC/HRMS)

NASA Astrophysics Data System (ADS)

Hu, Yuntao; Zheng, Qing; Noll, Lisa; Zhang, Shasha; Wanek, Wolfgang

2017-04-01

Organic nitrogen transformation processes are the key driver of soil nitrogen availability, strongly affecting the nitrogen turnover and carbon cycling of terrestrial ecosystems. Low molecular weight organic nitrogen compounds (e.g. amino acids and amino sugars) that can be directly utilized by plants or microorganisms are released by the extracellular cleavage of high molecular weight organic nitrogen compounds (e.g. proteins, peptidoglycan, and chitin) by hydrolytic enzymes. This decomposition process is believed to be the rate-limiting step in the soil N cycle. Direct measurements of the in situ transformation rates of these small N compounds is highly challenging but can be realized by applying the isotope pool dilution (IPD) technique, in which the target compound pool is labeled with isotopic tracers and subsequently the dilution of the tracers is measured. We have recently pioneered the development of IPD assays to investigate the in situ flux of proteinaceous amino acids and glucose due to decomposition of organic matter and microbial utilization, but the roles of fluxes of amino sugars and amino acid enantiomers in soil nitrogen transformation processes are still unknown due to the lack of feasible extraction, purification, separation and detection methods. Here we developed a 15N IPD assay by utilizing a novel LC/HRMS (Orbitrap) platform, with the aim to measure transformation rates of amino sugars and amino acid enantiomers. After the tracer experiments soil extracts were purified by solid phase extraction prior to the analysis by MS. The utilization of Orbitrap-HRMS allowed us to resolve the mass signals of unlabeled analytes, and their 15N labeled (tracers) and 13C labeled (internal standards) analogues. The commercially unavailable 15N and 13C labeled amino sugars and amino acid enantiomers were produced from bacterial cell walls after batch culture in labeled growth media. This workflow was validated with soils from two sampling sites, allowing us to successfully investigate the production and consumption of 2 amino sugars, 18 amino acids, and 4 amino acid enantiomers in soils. We further applied this method to soils from 6 sampling sites differing in geology and land management, after short-term (1-day) temperature (5˚ C, 15˚ C, 25˚ C) pre-incubations. We found that the release of amino sugars (free glucosamine) during the decomposition of peptidoglycan and chitin accounted for approximately 5% to 15% of the total influx into the dissolved organic nitrogen pool (amino acids plus amino sugars). Muramic acid exhibited significantly longer residence times in soils, indicating that free muramic acid was not an important decomposition product of peptidoglycan in soil. We will present further results on potential controls of soil amino sugar fluxes, such as soil temperature, geology and land management, as well as soil peptidoglycan and chitin content, hydrolytic enzyme activity, and microbial community structure. These findings and further ongoing work will greatly advance our knowledge of the transformation processes of soil organic nitrogen and its major controls.

Sewage derive [sup 15]N in the Baltic traced in fucus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hobbie, J.E.; Fry, B.; Larsson, U.

Himmerfjarden, a fjord-like bay on the eastern shore of the Baltic, receives treated sewage from 250,000 inhabitants. Because the inorganic N in the effluent is enriched in [sup 15]N through denitrification, nitrification, and ammonia volatilization, an analysis of the distribution of [sup 15]N in the Bay tells how far from the source the sewage nitrogen moves. The attached macroalga Fucus vesiculosus was collected in early May from rocky shore at 0-0.5 m depth and the [sup 15]N content of the tips of the fronds analyzed. This N represents uptake and storage during the previous six months and growth during Marchmore » and April. The [delta][sup 15]N was uniformly high (11-13[per thousand]) in the main body of the Bay within 15 km from the sewage source. Beyond 15 km values decreased with distance to a low of 4.6[per thousand] at 35 km, where the Bay ends and the coastal waters begin. Using the 11-13 and 4.6[per thousand] as endmembers, the percentage of sewage N making up the Fucus at any point may be calculated. The [delta][sub 15]N of particulate organic matter in the offshore Baltic waters was around 0[per thousand] and Fucus had an [delta][sup 15]N about 1.5[per thousand] higher than the POM. From this and other evidence we conclude that there is a belt of coastal water with an elevated [delta][sup 15]N lying along the east coast of the Baltic. This presumably derives from sewage and perhaps from agriculture and is potentially of use as a tracer of coastal zone/pelagic zone interactions.« less

16 CFR 300.15 - Labeling of containers or packaging of wool products.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Labeling of containers or packaging of wool products. 300.15 Section 300.15 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.15...

Nitrite fixation by humic substances: Nitrogen-15 nuclear magnetic resonance evidence for potential intermediates in chemodenitrification

USGS Publications Warehouse

Thorn, K.A.; Mikita, M.A.

2000-01-01

Studies have suggested that NO2/-, produced during nitrification and denitrification, can become incorporated into soil organic matter and, in one of the processes associated with chemodenitrification, react with organic matter to form trace N gases, including N2O. To gain an understanding of the nitrosation chemistry on a molecular level, soil and aquatic humic substances were reacted with 15N-labeled NaNO2, and analyzed by liquid phase 15N and 13C nuclear magnetic resonance (NMR). The International Humic Substances Society (IHSS) Pahokee peat and peat humic acid were also reacted with Na15NO2 and analyzed by solid-state 15N NMR. In Suwannee River, Armadale, and Laurentian fulvic acids, phenolic rings and activated methylene groups underwent nitrosation to form nitrosophenols (quinone monoximes) and ketoximes, respectively. The oximes underwent Beckmann rearrangements to 2??amides, and Beckmann fragmentations to nitriles. The nitriles in turn underwent hydrolysis to 1??amides. Peaks tentatively identified as imine, indophenol, or azoxybenzene nitrogens were clearly present in spectra of samples nitrosated at pH 6 but diminished at pH 3. The 15N NMR spectrum of the peat humic acid exhibited peaks corresponding with N-nitroso groups in addition to nitrosophenols, ketoximes, and secondary Beckmann reaction products. Formation of N-nitroso groups was more significant in the whole peat compared with the peat humic acid. Carbon-13 NMR analyses also indicated the occurrence of nitrosative demethoxylation in peat and soil humic acids. Reaction of 15N-NH3 fixated fulvic acid with unlabeled NO2/- resulted in nitrosative deamination of aminohydroquinone N, suggesting a previously unrecognized pathway for production of N2 gas in soils fertilized with NH3.Studies have suggested that NO2-, produced during nitrification and denitrification, can become incorporated into soil organic matter and, in one of the processes associated with chemodenitrification, react with organic matter to form trace N gases, including N2O. To gain an understanding of the nitrosation chemistry on a molecular level, soil and aquatic humic substances were reacted with 15N-labeled NaNO2, and analyzed by liquid phase 15N and 13C nuclear magnetic resonance (NMR). The International Humic Substances Society (IHSS) Pahokee peat and peat humic acid were also reacted with Na15NO2 and analyzed by solid-state 15N NMR. In Suwannee River, Armadale, and Laurentian fulvic acids, phenolic rings and activated methylene groups underwent nitrosation to form nitrosophenols (quinone monoximes) and ketoximes, respectively. The oximes underwent Beckmann rearrangements to 2?? amides, and Beckmann fragmentations to nitriles. The nitriles in turn underwent hydrolysis to 1?? amides. Peaks tentatively identified as imine, indophenol, or azoxybenzene nitrogens were dearly present in spectra of samples nitrosated at pH 6 but diminished at pH 3. The 15N NMR spectrum of the peat humic acid exhibited peaks corresponding with N-nitroso groups in addition to nitrosophenols, ketoximes, and secondary Beckmann reaction products. Formation of N-nitroso groups was more significant in the whole peat compared with the peat humic acid. Carbon-13 NMR analyses also indicated the occurrence of nitrosative demethoxylation in peat and soil humic acids. Reaction of 15N-NH3 fixated fulvic acids with unlabeled NO2- resulted in nitrosative deamination of aminohydroquinone N, suggesting a previously unrecognized pathway for production of N2 gas in soils fertilized with NH3.

Ultraviolet irradiation effects incorporation of nitrate and nitrite nitrogen into aquatic natural organic matter

USGS Publications Warehouse

Thorn, Kevin A.; Cox, Larry G.

2012-01-01

One of the concerns regarding the safety and efficacy of ultraviolet radiation for treatment of drinking water and wastewater is the fate of nitrate, particularly its photolysis to nitrite. In this study, 15N NMR was used to establish for the first time that UV irradiation effects the incorporation of nitrate and nitrite nitrogen into aquatic natural organic matter (NOM). Irradiation of 15N-labeled nitrate in aqueous solution with an unfiltered medium pressure mercury lamp resulted in the incorporation of nitrogen into Suwannee River NOM (SRNOM) via nitrosation and other reactions over a range of pH from approximately 3.2 to 8.0, both in the presence and absence of bicarbonate, confirming photonitrosation of the NOM. The major forms of the incorporated label include nitrosophenol, oxime/nitro, pyridine, nitrile, and amide nitrogens. Natural organic matter also catalyzed the reduction of nitrate to ammonia on irradiation. The nitrosophenol and oxime/nitro nitrogens were found to be susceptible to photodegradation on further irradiation when nitrate was removed from the system. At pH 7.5, unfiltered irradiation resulted in the incorporation of 15N-labeled nitrite into SRNOM in the form of amide, nitrile, and pyridine nitrogen. In the presence of bicarbonate at pH 7.4, Pyrex filtered (cutoff below 290–300 nm) irradiation also effected incorporation of nitrite into SRNOM as amide nitrogen. We speculate that nitrosation of NOM from the UV irradiation of nitrate also leads to production of nitrogen gas and nitrous oxide, a process that may be termed photo-chemodenitrification. Irradiation of SRNOM alone resulted in transformation or loss of naturally abundant heterocyclic nitrogens.

DISTRIBUTION OF RADIOACTIVITY IN AUTOLYZED CELL WALL OF BACILLUS CEREUS DURING SPHEROPLAST FORMATION1

PubMed Central

Kronish, Donald P.; Mohan, Raam R.; Schwartz, Benjamin S.

1964-01-01

Kronish, Donald P. (Warner-Lambert Research Institute, Morris Plains, N.J.), Raam R. Mohan, and Benjamin S. Schwartz. Distribution of radioactivity in autolyzed cell wall of Bacillus cereus during spheroplast formation. J. Bacteriol. 87:581–587. 1964.—Spheroplasts of Bacillus cereus strain T were produced from cells grown in the presence of uniformly labeled C14-glucose. At regular intervals during spheroplast formation, enzymatically degraded cell wall was isolated by a new procedure. Radioactivity of solubilized cell wall in cell-free material increased from 2.5 to 42% of the total incorporated label during spheroplast formation. The rate of cell-wall degradation as measured by increase in radioactivity was biphasic with relative slopes of 2.0 and 5.0. During autolytic depolymerization of B. cereus cell wall, two major components were solubilized at different rates. Chemical fractionation revealed these to be a peptide and a mucopeptide. The possibility of two enzymes being involved in spheroplast formation and cell-wall degradation is discussed. Images PMID:14127573

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1988-01-01

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

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

PubMed Central

Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.

2008-01-01

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins. PMID:18005930

Improvement of 19F MR image uniformity in a mouse model of cellular therapy using inductive coupling.

PubMed

Park, Bu S; Ma, Ge; Koch, William T; Rajan, Sunder S; Mastromanolis, Manuel; Lam, Johnny; Sung, Kyung; McCright, Brent

2018-06-15

Improve 19 F magnetic resonance imaging uniformity of perfluorocarbon (PFC)-labeled cells by using a secondary inductive resonator tuned to 287 MHz to enhance the induced radio frequency (RF) magnetic field (B 1 ) at 7.05 T. Following Faraday's induction law, the sign of induced B 1 made by the secondary resonator can be changed depending on the tuning of the resonator. A secondary resonator located on the opposite side of the phantom of the 19 F surface coil can be shown to enhance or subtract the induced B 1 field, depending upon its tuning. The numerical simulation results of rotating transmit B 1 magnitude (|B 1 + |) and corresponding experimental 19 F images were compared without and with the secondary resonator. With the secondary resonator tuned to 287 MHz, improvements of |B 1 + | and 19 F image uniformity were demonstrated. The use of the secondary resonator improved our ability to visualize transplanted cell location non-invasively over a period of 6 weeks. The secondary resonator tuned to enhance the induced B 1 results in improved image uniformity in a pre-clinical application, enabling cell tracking of PFC-labeled cells with the secondary resonator.

Fate of microbial nitrogen, carbon, hydrolysable amino acids, monosaccharides, and fatty acids in sediment

NASA Astrophysics Data System (ADS)

Veuger, Bart; van Oevelen, Dick; Middelburg, Jack J.

2012-04-01

The fate of microbial carbon, nitrogen, hydrolysable amino acids (HAAs), monosaccharides, and fatty acids in sediment was investigated experimentally. The microbial community of a tidal flat sediment was labeled with 13C-enriched glucose and 15N-enriched ammonium, and sediment was incubated for up to 371 days. Analysis of total concentrations and 13C- and 15N content of bulk sediment, hydrolysable amino acids (including D-alanine), monosaccharides, total fatty acids (TFAs), and phospholipid-derived fatty acids (PLFAs) allowed us to trace the fate of microbial biomass and -detritus and the major biochemical groups therein (proteins, carbohydrates, and lipids) over intermediate time scales (weeks-months). Moreover, the unidentified fraction of the labeled material (i.e. not analyzed as HAA, FA, or carbohydrate) provided information on the formation and fate of molecularly uncharacterizable organic matter. Loss of 13C and 15N from the sediment was slow (half live of 433 days) which may have been due to the permanently anoxic conditions in the experiment. Loss rates for the different biochemical groups were also low with the following order of loss rate constants: PLFA > TFA > HAA > monosaccharides. The unidentified 13C-pool was rapidly formed (within days) and then decreased relatively slowly, resulting in a gradual relative accumulation of this pool over time. Degradation and microbial reworking of the labeled material resulted in subtle, yet consistent, diagenetic changes within the different biochemical groups. In the HAA pool, glycine, lysine, and proline were lost relatively slowly (i.e. best preserved) while there was no accumulation of D-alanine relative to L-alanine, indicating no relative accumulation of bacterial macromolecules rich in D-alanine. In the fatty acid pool, there was very little difference between PLFAs and TFAs, indicating a very similar lability of these pools. Differences between individual fatty acids included a relatively slow loss of i15:0, ai15:0 and 18:1ω7c which likely resulted from production of these bacterial fatty acids during bacterial reworking of the organic matter. Differences between loss rate constants for individual monosaccharides were not significant. An exception was ribose that was produced and lost relatively rapidly, which may be related to ribose being an important component of RNA. Losses of bulk 13C and 15N were closely coupled despite partly being present in different biochemicals and partly being derived from different microbial sources, indicating no selective preservation of either C or N during organic matter diagenesis.

16 CFR Figures 14 and 15 to Part 1633 - Label for Domestic Mattress Alone and with Foundation and Label for Imported Mattress Alone and...

Code of Federal Regulations, 2010 CFR

2010-01-01

... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Label for Domestic Mattress Alone and with Foundation and Label for Imported Mattress Alone and With Foundation 14 Figures 14 and 15 to Part 1633... FLAMMABILITY (OPEN FLAME) OF MATTRESS SETS Pt.1633, Figs. 14, 15 Figures 14 and 15 to Part 1633—Label for...

16 CFR Figures 14 and 15 to Part 1633 - Label for Domestic Mattress Alone and with Foundation and Label for Imported Mattress Alone and...

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Label for Domestic Mattress Alone and with Foundation and Label for Imported Mattress Alone and With Foundation 14 Figures 14 and 15 to Part 1633... FLAMMABILITY (OPEN FLAME) OF MATTRESS SETS Pt.1633, Figs. 14, 15 Figures 14 and 15 to Part 1633—Label for...

Isotope-Labeled Composition B for Tracing Detonation Signatures

NASA Astrophysics Data System (ADS)

Manner, Virginia; Podlesak, David; Huber, Rachel; Amato, Ronald; Giambra, Anna; Bowden, Patrick; Hartline, Ernest; Dattelbaum, Dana

2017-06-01

To better understand how solid carbon forms and evolves during detonation, we have prepared Composition B with 13 C and 15 N-labeled 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) and 2,4,6-trinitrotoluene (TNT) in order to trace the formation of soot from the carbon and nitrogen atoms in these explosives. Isotope-labeling of explosives has been performed in the recent past for a variety of reasons, including environmental remediation and reaction mechanism studies. Because it is expensive and time consuming to prepare these materials, and our detection equipment only requires trace amounts of isotopes, we have prepared fully-labeled materials and substituted them into unlabeled RDX and TNT at less than the 1% level. We will discuss the preparation and full characterization of this labeled Composition B, the detonation tests performed, along with the results of the post-detonation soot analysis. Various detonation models predict differing amounts and forms of carbon and nitrogen; these isotopically-labeled precursors have allowed these models to be tested.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Renslow, Ryan S.; Lindemann, Stephen R.; Cole, Jessica K.

EElucidating nutrient exchange in microbial communities is an important step in understanding the relationships between microbial systems and global biogeochemical cycles, but these communities are complex and the interspecies interactions that occur within them are not well understood. Phototrophic consortia are useful and relevant experimental systems to investigate such interactions as they are not only prevalent in the environment, but some are cultivable in vivo and amenable to controlled scientific experimentation. High spatial resolution secondary ion mass spectrometry (NanoSIMS) is a powerful tool capable of visualizing the metabolic activities of single cells within a biofilm, but quantitative analysis of themore » resulting data has typically been a manual process, resulting in a task that is both laborious and susceptible to human error. Here, we describe the creation and application of a semi-automated image-processing pipeline that can analyze NanoSIMS-generated data of phototrophic biofilms. The tool employs an image analysis process, which includes both elemental and morphological segmentation, producing a final segmented image that allows for discrimination between autotrophic and heterotrophic biomass, the detection of individual cyanobacterial filaments and heterotrophic cells, the quantification of isotopic incorporation of individual heterotrophic cells, and calculation of relevant population statistics. We demonstrate the functionality of the tool by using it to analyze the uptake of 15N provided as either nitrate or ammonium through the unicyanobacterial consortium UCC-O and imaged via NanoSIMS. We found that the degree of 15N incorporation by individual cells was highly variable when labeled with 15NH4 +, but much more even when biofilms were labeled with 15NO3-. In the 15NH4 +-amended biofilms, the heterotrophic distribution of 15N incorporation was highly skewed, with a large population showing moderate 15N incorporation and a small number of organisms displaying very high 15N uptake. The results showed that analysis of NanoSIMS data can be performed in a way that allows for quantitation of the elemental uptake of individual cells, a technique necessary for advancing research into the metabolic networks that exist within biofilms with statistical analyses that are supported by automated, user-friendly processes.« less

[Accumulation responses of seeds and seedlings to 15N isotope for two typical broadleaved trees in Northeast China.

PubMed

Wang, Guang Chen; Song, Yuan; Yan, Qiao Ling; Zhang, Jin Xin

2016-08-01

Two typical broadleaved trees (i.e., Fraxinus rhynchophylla and Acer mono) with wind-dispersed seeds in Northeast China were selected in this study. A method of 15 N isotope labeling was used to explore the accumulation responses of seeds and seedlings to 15 N-urea soaking concentration (0, 0.05, 0.1 and 0.2 g·L -1 ), soaking time (4, 8 and 12 days) and leaf stage (2, 4, 6, and 8 leaves). The results showed that 15 N-urea soaking concentration and soaking time had significantly positive effects on δ 15 N values of seeds, i.e., higher 15 N-urea concentration and longer period of soaking (0.2 g·L -1 +12 d) were contributed to more 15 N accumulation of seeds. The maximum multiples of 15 N accumulation in F. rhynchophylla seeds and A. mono seeds were observed in 0.1 g·L -1 + (4 d, 8 d) and 0.05 g·L -1 + (4 d, 8 d), respectively. The loss rate of δ 15 N values decreased markedly from 2 leaves to 6 leaves and then kept relatively stable with the increasing seedling height, and the total δ 15 N values of seedlings started to decline at the stage of 8 leaves. These results suggested that seedlings with 6 leaves were more suitable for tracking seedling source. The δ 15 N values in leaves of seedlings were significantly positively correlated with 15 N-urea concentration level, soaking time and δ 15 N values of seeds. Overall, the accumulation of 15 N-urea could be found in seeds and seedlings of F. rhynchophylla and A. mono. The combination of 15 N-urea concentration (0.1 g·L -1 ), soaking time (8 d) and leaf stage (6 leaves) was the most suitable for tracking the seeds and seedlings of these two broadleaved trees.

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

NASA Astrophysics Data System (ADS)

Kanamori, Keiko; Ross, Brian D.

2001-12-01

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

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

PubMed

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

2016-06-01

The formation and stabilization of soil organic matter (SOM) are major concerns in the context of global change for carbon sequestration and soil health. It is presently believed that lignin is not selectively preserved in soil and that chemically labile compounds bonding to minerals comprise a large fraction of the SOM. Labile plant inputs have been suggested to be the main precursor of the mineral-bonded SOM. Litter decomposition and SOM formation are expected to have temperature sensitivity varying with the lability of plant inputs. We tested this framework using dual (13) C and (15) N differentially labeled plant material to distinguish the metabolic and structural components within a single plant material. Big Bluestem (Andropogon gerardii) seedlings were grown in an enriched (13) C and (15) N environment and then prior to harvest, removed from the enriched environment and allowed to incorporate natural abundance (13) C-CO2 and (15) N fertilizer into the metabolic plant components. This enabled us to achieve a greater than one atom % difference in (13) C between the metabolic and structural components within the plant litter. This differentially labeled litter was incubated in soil at 15 and 35 °C, for 386 days with CO2 measured throughout the incubation. After 14, 28, 147, and 386 days of incubation, the soil was subsequently fractionated. There was no difference in temperature sensitivity of the metabolic and structural components with regard to how much was respired or in the amount of litter biomass stabilized. Only the metabolic litter component was found in the sand, silt, or clay fraction while the structural component was exclusively found in the light fraction. These results support the stabilization framework that labile plant components are the main precursor of mineral-associated organic matter. © 2016 John Wiley & Sons Ltd.

U.S. EPA, Pesticide Product Label, , 07/26/1989

EPA Pesticide Factsheets

2011-04-21

... 'I.sb " • .,11, ... Il.".,li, .it. cl •• r MIler f., .t leist 15 1.1111'". I_~iattl, cnhct ,l,.icin. h cu •• , I.,oslin. i_~i.ttl, cnhct ",sieia .... , i .. II,Ui., " .. u It II n"lScin. ...

Labile dissolved organic carbon supply limits hyporheic denitrification

Treesearch

Jay P. Zarnetske; Roy Haggerty; Steven M. Wondzell; Michelle A. Baker

2012-01-01

We used an in situ steady state 15N-labeled nitrate and acetate well-to-wells injection experiment to determine how the availability of labile dissolved organic carbon as acetate influences microbial denitrification in the hyporheic zone of an upland (third-order) agricultural stream.

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

NASA Astrophysics Data System (ADS)

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

2017-04-01

The suitability of Siberia for agriculture is expected to increase in the next decades due to strong and rapid climatic changes, but little is known on the environmental drivers of soil fertility there, especially nitrogen (N). Plant-available N is mainly derived from litter decomposition. South-western (SW) Siberia is located on the transition between several bioclimatic zones that are predicted to shift and extend along with climate change (steppe, forest-steppe, sub-taiga). The soils of this region are formed on a common loess deposit but they are submitted to different climatic conditions and vegetation cover. In the south of the region, typically in steppe/forest-steppe, soil freezes over winter because of a relatively shallow snow-pack, and water shortages are frequent in summer. In the north, typically in sub-taiga, the soil is barely frozen in winter due to a thick snow-pack and sufficient soil moisture in summer. In this study, we characterized the dynamics of leaf litter decomposition and the transfer of N from leaf litter to the soil and back to plants. Four sites were chosen along a climate gradient (temperature, precipitation and snow depth). At each site, we applied 15N-labelled leaf litter on the soil surface in experimental plots in an aspen (Populus tremula L.) forest and in a grassland. Twice a year during three years, we tracked the 15N derived from the decomposing labelled-litter in the organic layers, in the first 15 cm of the soil, and in above-ground vegetation. Soil temperature and moisture were monitored at a daily timestep over three years and soil water budgets were simulated (BILJOU model, Granier et al. 1999). We observed contrasting patterns in the fate of litter-derived 15N between bioclimatic zones. Over three years, along with faster decay rates, the release of leaf litter-N was faster in sub-taiga than in forest-steppe. As such, higher quantities of 15N were transferred into the soil in sub-taiga. The transfer was also deeper there, which might be related to a more intense drainage because of higher snow levels, as inferred from soil water budget modelling. Interestingly, this higher drainage seems to induce only a small loss of N from the system. Such retention could result from soil physico-chemical properties (higher fine silt and oxides contents) enhancing soil organic matter stabilization, and/or by the immobilization of N in microbial metabolites. We observed differing N dynamics between forest and grassland that can be related to the different chemical composition of initial litter (tree leaves vs. grasses) and plant-soil interactions. In general, N was retained in the first centimeters of the mineral soil in grassland while the transfer was deeper in the forest soils. As fine root exploration is denser in grassland topsoil than in forest topsoil, we infer that an efficient uptake of N by grasses in the first soil layers limits N migration down the profile. It is also possible that grasses are active earlier in the season than trees and understorey species, i.e. at snow-melt when drainage is the most intense.

Metabolic effect of TAp63α: enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense

PubMed Central

D'Alessandro, Angelo; Amelio, Ivano; Berkers, Celia R.; Antonov, Alexey; Vousden, Karen H.; Melino, Gerry; Zolla, Lello

2014-01-01

TAp63α is a member of the p53 family, which plays a central role in epithelial cancers. Recently, a role has emerged for p53 family members in cancer metabolic modulation. In order to assess whether TAp63α plays a role in cancer metabolism, we exploited p53-null osteosarcoma Tet-On Saos-2 cells, in which the expression of TAp63α was dependent on doxycycline supplementation to the medium. Metabolomics labeling experiments were performed by incubating the cells in 13C-glucose or 13C15N-glutamine-labeled culture media, as to monitor metabolic fluxes upon induced expression of TAp63α. Induced expression of TAp63α resulted in cell cycle arrest at the G1 phase. From a metabolic standpoint, expression of Tap63α promoted glycolysis and the pentose phosphate pathway, which was uncoupled from nucleotide biosynthesis, albeit prevented oxidative stress in the form of oxidized glutathione. Double 13C-glucose and 13C15N-glutamine metabolic labeling confirmed that induced expression of TAp63α corresponded to a decreased flux of pyruvate to the Krebs cycle and decreased utilization of glutamine for catabolic purposes in the TCA cycle. Results were not conclusive in relation to anabolic utilization of labeled glutamine, since it is unclear to what extent the observed minor TAp63α-dependent increases of glutamine-derived labeling in palmitate could be tied to increased rates of reductive carboxylation and de novo synthesis of fatty acids. Finally, bioinformatics elaborations highlighted a link between patient survival rates and the co-expression of p63 and rate limiting enzymes of the pentose phosphate pathway, G6PD and PGD. PMID:25229745

Attenuation by genistein of sodium-chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

PubMed

Tatsuta, M; Iishi, H; Baba, M; Yano, H; Uehara, H; Nakaizumi, A

1999-01-29

The effects of prolonged administration of genistein, a tyrosine-kinase inhibitor, on sodium-chloride-enhanced induction of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine, and the labeling and apoptotic indices and vessel counts in the gastric mucosa and gastric cancers, were investigated in Wistar rats. After 25 weeks of the carcinogen treatment, rats were fed chow pellets containing 10% sodium chloride and were given s.c. injections of genistein at dosages of 15 mg/kg or 30 mg/kg body weight every other day. In week 52, the incidence of gastric cancers was significantly greater in rats fed sodium chloride than in untreated control rats. Prolonged administration of genistein at a dosage of 30 mg/kg, but not 15 mg/kg, body weight significantly reduced the incidence of gastric cancers, which was increased by oral treatment with sodium chloride. Genistein at the higher dose significantly decreased the labeling index and vessel counts of the antral mucosa and the gastric cancers (which were increased by treatment with sodium chloride) and significantly increased the apoptotic index of the antral mucosa and the cancers (which was lowered by the treatment with sodium chloride). These findings suggest that genistein attenuates gastric carcinogenesis promoted by sodium chloride, by inducing increased apoptosis and lower cell proliferation and angiogenesis of antral mucosa and gastric cancers.

Global Trophic Position Comparison of Two Dominant Mesopelagic Fish Families (Myctophidae, Stomiidae) Using Amino Acid Nitrogen Isotopic Analyses

PubMed Central

Choy, C. Anela; Davison, Peter C.; Drazen, Jeffrey C.; Flynn, Adrian; Gier, Elizabeth J.; Hoffman, Joel C.; McClain-Counts, Jennifer P.; Miller, Todd W.; Popp, Brian N.; Ross, Steve W.; Sutton, Tracey T.

2012-01-01

The δ15N values of organisms are commonly used across diverse ecosystems to estimate trophic position and infer trophic connectivity. We undertook a novel cross-basin comparison of trophic position in two ecologically well-characterized and different groups of dominant mid-water fish consumers using amino acid nitrogen isotope compositions. We found that trophic positions estimated from the δ15N values of individual amino acids are nearly uniform within both families of these fishes across five global regions despite great variability in bulk tissue δ15N values. Regional differences in the δ15N values of phenylalanine confirmed that bulk tissue δ15N values reflect region-specific water mass biogeochemistry controlling δ15N values at the base of the food web. Trophic positions calculated from amino acid isotopic analyses (AA-TP) for lanternfishes (family Myctophidae) (AA-TP ∼2.9) largely align with expectations from stomach content studies (TP ∼3.2), while AA-TPs for dragonfishes (family Stomiidae) (AA-TP ∼3.2) were lower than TPs derived from stomach content studies (TP∼4.1). We demonstrate that amino acid nitrogen isotope analysis can overcome shortcomings of bulk tissue isotope analysis across biogeochemically distinct systems to provide globally comparative information regarding marine food web structure. PMID:23209656

Global trophic position comparison of two dominant mesopelagic fish families (Myctophidae, Stomiidae) using amino acid nitrogen isotopic analyses

USGS Publications Warehouse

Choy, C. Anela; Davison, Peter C.; Drazen, Jeffrey C.; Flynn, Adrian; Gier, Elizabeth J.; Hoffman, Joel C.; McClain-Counts, Jennifer P.; Miller, Todd W.; Popp, Brian N.; Ross, Steve W.; Sutton, Tracey T.

2012-01-01

The δ15N values of organisms are commonly used across diverse ecosystems to estimate trophic position and infer trophic connectivity. We undertook a novel cross-basin comparison of trophic position in two ecologically well-characterized and different groups of dominant mid-water fish consumers using amino acid nitrogen isotope compositions. We found that trophic positions estimated from the δ15N values of individual amino acids are nearly uniform within both families of these fishes across five global regions despite great variability in bulk tissue δ15N values. Regional differences in the δ15N values of phenylalanine confirmed that bulk tissue δ15N values reflect region-specific water mass biogeochemistry controlling δ15N values at the base of the food web. Trophic positions calculated from amino acid isotopic analyses (AA-TP) for lanternfishes (family Myctophidae) (AA-TP ~2.9) largely align with expectations from stomach content studies (TP ~3.2), while AA-TPs for dragonfishes (family Stomiidae) (AA-TP ~3.2) were lower than TPs derived from stomach content studies (TP~4.1). We demonstrate that amino acid nitrogen isotope analysis can overcome shortcomings of bulk tissue isotope analysis across biogeochemically distinct systems to provide globally comparative information regarding marine food web structure.

An Upgrade Pinning Block: A Mechanical Practical Aid for Fast Labelling of the Insect Specimens.

PubMed

Ghafouri Moghaddam, Mohammad Hossein; Ghafouri Moghaddam, Mostafa; Rakhshani, Ehsan; Mokhtari, Azizollah

2017-01-01

A new mechanical innovation is described to deal with standard labelling of dried specimens on triangular cards and/or pinned specimens in personal and public collections. It works quickly, precisely, and easily and is very useful for maintaining label uniformity in collections. The tools accurately sets the position of labels in the shortest possible time. This tools has advantages including rapid processing, cost effectiveness, light weight, and high accuracy, compared to conventional methods. It is fully customisable, compact, and does not require specialist equipment to assemble. Conventional methods generally require locating holes on the pinning block surface when labelling with a resulting risk to damage of the specimens. Insects of different orders can be labelled by this simple and effective tool.

An Upgrade Pinning Block: A Mechanical Practical Aid for Fast Labelling of the Insect Specimens

PubMed Central

Ghafouri Moghaddam, Mohammad Hossein; Rakhshani, Ehsan; Mokhtari, Azizollah

2017-01-01

Abstract A new mechanical innovation is described to deal with standard labelling of dried specimens on triangular cards and/or pinned specimens in personal and public collections. It works quickly, precisely, and easily and is very useful for maintaining label uniformity in collections. The tools accurately sets the position of labels in the shortest possible time. This tools has advantages including rapid processing, cost effectiveness, light weight, and high accuracy, compared to conventional methods. It is fully customisable, compact, and does not require specialist equipment to assemble. Conventional methods generally require locating holes on the pinning block surface when labelling with a resulting risk to damage of the specimens. Insects of different orders can be labelled by this simple and effective tool. PMID:29104440

Effects of positive expiratory pressure on pulmonary clearance of aerosolized technetium-99m-labeled diethylenetriaminepentaacetic acid in healthy individuals

PubMed Central

de Albuquerque, Isabella Martins; Cardoso, Dannuey Machado; Masiero, Paulo Ricardo; Paiva, Dulciane Nunes; Resqueti, Vanessa Regiane; Fregonezi, Guilherme Augusto de Freitas; Menna-Barreto, Sérgio Saldanha

2016-01-01

ABSTRACT Objective: To evaluate the effects of positive expiratory pressure (PEP) on pulmonary epithelial membrane permeability in healthy subjects. Methods: We evaluated a cohort of 30 healthy subjects (15 males and 15 females) with a mean age of 28.3 ± 5.4 years, a mean FEV1/FVC ratio of 0.89 ± 0.14, and a mean FEV1 of 98.5 ± 13.1% of predicted. Subjects underwent technetium-99m-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA) radioaerosol inhalation lung scintigraphy in two stages: during spontaneous breathing; and while breathing through a PEP mask at one of three PEP levels-10 cmH2O (n = 10), 15 cmH2O (n = 10), and 20 cmH2O (n = 10). The 99mTc-DTPA was nebulized for 3 min, and its clearance was recorded by scintigraphy over a 30-min period during spontaneous breathing and over a 30-min period during breathing through a PEP mask. Results: The pulmonary clearance of 99mTc-DTPA was significantly shorter when PEP was applied-at 10 cmH2O (p = 0.044), 15 cmH2O (p = 0.044), and 20 cmH2O (p = 0.004)-in comparison with that observed during spontaneous breathing. Conclusions: Our findings indicate that PEP, at the levels tested, is able to induce an increase in pulmonary epithelial membrane permeability and lung volume in healthy subjects. PMID:28117469

A novel method to label preformed liposomes with 64Cu for positron emission tomography (PET) imaging.

PubMed

Seo, Jai Woong; Zhang, Hua; Kukis, David L; Meares, Claude F; Ferrara, Katherine W

2008-12-01

Radiolabeling of liposomes with 64Cu (t(1/2)=12.7 h) is attractive for molecular imaging and monitoring drug delivery. A simple chelation procedure, performed at a low temperature and under mild conditions, is required to radiolabel preloaded liposomes without lipid hydrolysis or the release of the encapsulated contents. Here, we report a 64Cu postlabeling method for liposomes. A 64Cu-specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT), was conjugated with an artificial lipid to form a BAT-PEG-lipid. After incorporation of 0.5% (mol/mol) BAT-PEG-lipid during liposome formulation, liposomes were successfully labeled with 64Cu in 0.1 M NH4OAc pH 5 buffer at 35 degrees C for 30-40 min with an incorporation yield as high as 95%. After 48 h of incubation of 64Cu-liposomes in 50/50 serum/PBS solution, more than 88% of the 64Cu label was still associated with liposomes. After injection of liposomal 64Cu in a mouse model, 44+/-6.9, 21+/-2.7, 15+/-2.5, and 7.4+/-1.1 (n=4) % of the injected dose per cubic centimeter remained within the blood pool at 30 min, 18, 28, and 48 h, respectively. The biodistribution at 48 h after injection verified that 7.0+/-0.47 (n=4) and 1.4+/-0.58 (n=3) % of the injected dose per gram of liposomal 64Cu and free 64Cu remained in the blood pool, respectively. Our results suggest that this fast and easy 64Cu labeling of liposomes could be exploited in tracking liposomes in vivo for medical imaging and targeted delivery.

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

PubMed

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

2014-11-01

Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production. © The Author 2013. Published by Oxford University Press.

Inorganic and organic nitrogen uptake by phytoplankton and heterotrophic bacteria in the stratified Mid-Atlantic Bight

NASA Astrophysics Data System (ADS)

Bradley, Paul B.; Sanderson, Marta P.; Frischer, Marc E.; Brofft, Jennifer; Booth, Melissa G.; Kerkhof, Lee J.; Bronk, Deborah A.

2010-08-01

Little is known about the relative importance of inorganic and organic nitrogen (N) sources in fueling production of phytoplankton versus heterotrophic bacteria on the continental shelf. This issue was addressed during two diel experiments conducted in the Mid-Atlantic Bight at the Long-term Ecosystem Observatory, LEO-15, off southern New Jersey. Uptake of 15N-labeled ammonium (NH 4+), nitrate (NO 3-), and nitrite (NO 2-), and dual-labeled ( 15N and 13C) urea and dissolved free amino acids was measured in water taken from the surface and bottom mixed layers approximately every 4 h over two 24 h periods in July 2002. Two methods were used to quantify 15N uptake rates: (1) traditional filtration into various phytoplankton and bacterial size classes, and (2) flow cytometric (FCM) sorting of autotrophic cells based on the presence of chlorophyll autofluorescence. Due to a strong pycnocline, the nutrient composition was quite distinct between the surface and bottom mixed layers. Dissolved organic N (DON) comprised >99% of the total dissolved N (TDN) pool in surface waters, whereas the bottom-water TDN pool was roughly divided between NH 4+, NO 3-, and DON. Urea was the dominant N form used by all fractions at the surface, and although phytoplankton >3 μm was responsible for most of the urea uptake, bacterial use was detected using stable isotopes and also suggested by ureC sequence analysis. The majority of ureC sequences recovered from the 0.2-0.8 μm fraction belonged to members of the Alphaproteobacteria (46%), whereas those of the 0.8-3.0 μm size class consisted primarily of Cyanobacteria (70%). In contrast to the surface, N uptake in the bottom layer was dominated by NH 4+. The bacterial fraction was responsible for 20-49% of the size-fractionated NH 4+ and NO 3- uptake in surface samples and 36-93% at the bottom. These results suggest that organic N, such as urea, is a viable source of N nutrition to phytoplankton forced to compete with heterotrophic bacteria for limited inorganic N.

Use of levetiracetam in a population of patients aged 65 years and older: a subset analysis of the KEEPER trial.

PubMed

Ferrendelli, J A; French, J; Leppik, I; Morrell, M J; Herbeuval, A; Han, J; Magnus, L

2003-12-01

Levetiracetam (Keppra) was evaluated in a subset of patients aged >/=65 years (n=78) enrolled in a large (n=1030) open-label, phase IV trial (the KEEPER trial). A 4-week dose adjustment was followed by a 12-week evaluation period. An overall median reduction in partial seizures of 80.1% (n=65) was observed. Overall, 76.9% of patients were >/=50% responders, 56.9% were >/=75% responders, and 40.0% were 100% responders. Levetiracetam was well tolerated, with 42.3% of patients reporting one or more adverse events. A total of 15 patients (19.2%) experienced an adverse event that led to discontinuation. Somnolence (n=13,16.7%) and dizziness (n=7,9.0%) were the most commonly reported adverse events. Despite the limitations of the open-label study design, these data provide information regarding the use of levetiracetam as add-on therapy for the treatment of partial-onset seizures in patients >/=65 years of age, including those requiring concomitant medications.

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hastings, Adam F.; Otting, Gottfried; Folmer, Rutger H.A.

2005-09-23

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in {sup 1}H-{sup 15}N correlation spectra of a {sup 15}N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the {sup 1}H-{sup 15}N correlations was achieved using a suite of triple resonance NMR experiments with {sup 15}N,{sup 13}C,70% {sup 2}H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbationsmore » to {sup 1}H-{sup 15}N spectra revealed that the N-termini, {alpha}3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.« less

Mather AFB, California. Revised Uniform Summary of Surface Weather Observations. Parts A-F.

DTIC Science & Technology

1987-06-15

8217:A 3 513 MATHER 4FB :ALIFOPNIA REIurED UNIFORM IIMMARY ’,F TECN: AL APPLI:AT I 3KN CENTiiR CC A 15 UN37 N: :F ED U:AFC:.A: D:-37 042 F -,4/2 MI. L3A...cr : 2 a IA 4 0. 3 2 0 m W X 0 3r at 24- 4A or0 z 49 o- K6 aL 4 .l vi U, t 4 62 K KA =, -J4 eK 0.0 9k 0 0 ,4 K U, U, -r cc Li La a.. 2 64. 0 x 4 cc " A...N*. 0 0 0 0 0 a, 0 * 00 * N. * 00 * z 11 cr al z -a 0P 03 . 0 . n f-0 M * 0 via f- OM ~ -4 A . mf LA 0 1 0 6A0 *0 li "Z .0 0 0 0 L. 4L 0 .~ 0 -j U

49 CFR 172.411 - EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 2 2011-10-01 2011-10-01 false EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels..., EMERGENCY RESPONSE INFORMATION, TRAINING REQUIREMENTS, AND SECURITY PLANS Labeling § 172.411 EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label. (a) Except for size and color...

Wedge-shaped slice-selective adiabatic inversion pulse for controlling temporal width of bolus in pulsed arterial spin labeling

PubMed Central

Guo, Jia; Buxton, Richard B.; Wong, Eric C.

2015-01-01

Purpose In pulsed arterial spin labeling (PASL) methods, arterial blood is labeled via inverting a slab with uniform thickness, resulting in different temporal widths of boluses in vessels with different flow velocities. This limits the temporal resolution and signal-to-noise ratio (SNR) efficiency gains in PASL-based methods intended for high temporal resolution and SNR efficiency, such as Turbo-ASL and Turbo-QUASAR. Theory and Methods A novel wedge-shaped (WS) adiabatic inversion pulse is developed by adding in-plane gradient pulses to a slice-selective (SS) adiabatic inversion pulse to linearly modulate the inversion thicknesses at different locations while maintaining the adiabatic properties of the original pulse. A hyperbolic secant (HS) based WS inversion pulse was implemented. Its performance was tested in simulations, phantom and human experiments, and compared to an SS HS inversion pulse. Results Compared to the SS inversion pulse, the WS inversion pulse is capable of inducing different inversion thicknesses at different locations. It can be adjusted to generate a uniform temporal width of boluses in arteries at locations with different flow velocities. Conclusion The WS inversion pulse can be used to control the temporal widths of labeled boluses in PASL experiments. This should benefit PASL experiments by maximizing labeling duty cycle, and improving temporal resolution and SNR efficiency. PMID:26451521

Comparison between various densities of pore titanium meshes and e-polytetrafluoroethylene (ePTFE) membrane regarding bone regeneration induced by low intensity pulsed ultrasound (LIPUS) in rabbit nasal bone.

PubMed

Higuchi, Masatoshi; Moroi, Akinori; Yoshizawa, Kunio; Kosaka, Akihiko; Ikawa, Hiroumi; Iguchi, Ran; Saida, Yuriko; Hotta, Asami; Tsutsui, Takamitsu; Ueki, Koichiro

2016-09-01

The purpose of this study was to compare bone regenerative capability following use of polytetrafluoroethylene (ePTFE) membrane against that when various densities of pore titanium meshes are used with and without low intensity pulsed ultrasound (LIPUS). Adult male white rabbits were divided into 8 groups. In 4 groups, after incising along the nasal bone, four 3 × 8 mm bone defects were made in both sides and covered by an ePTFE membrane (group E: n = 15), a high density pore titanium mesh (group H: n = 15), a low density pore titanium mesh (group L: n = 15), and no mesh (control) (group C: n = 15). Furthermore, LIPUS was irradiated after surgery in 4 groups (groups EL, HL, LL and CL, in each n = 15). The rabbits were sacrificed at 1, 2 and 8 weeks postoperative, and formalin-fixed specimens were embedded in acrylic resin. The specimens were stained with hematoxylin and eosin. For immunohistochemical analysis, the specimens were treated with bone morphogenetic protein (BMP)-2 antibody. Group H had significantly higher values than groups L, E, and C regarding bone area ratio and labeling index of BMP-2 positive cells (P < 0.05). Furthermore, Group HL also had significantly higher values than the other groups regarding bone area ratio and labeling index of BMP-2 positive cells at 1, 2 and 8 weeks postoperative (P < 0.05). The results suggested that high density pore titanium mesh could induce new bone regeneration more than low density pore titanium mesh and ePTFE membrane. New bone formation may increase following LIPUS application. Copyright © 2016 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Silver Uptake, Distribution, and Effect on Calcium, Phosphorus, and Sulfur Uptake 1

PubMed Central

Koontz, Harold V.; Berle, Karen L.

1980-01-01

Bean, corn, and tomato plants were grown in a nutrient solution labeled with 32P, 45Ca, or 35S and varying concentrations of AgNO3. Following a 6-hour treatment period, plants were harvested and analyzed. A low Ag+ concentration (50 nanomolar) inhibited the shoot uptake of the ions investigated. In the roots, Ca uptake increased whereas P and S uptake decreased. Autoradiograms of bean and corn plants, using 110mAg, showed that Ag+ was uniformly deposited in the bean shoot, but corn shoots had regions of high activity along the leaf margins and at the tips where guttation had occurred. Roots were heavily labeled and shoots (especially the new growth) continued to accumulate Ag+ even after the intact plant was returned to Ag-free solution. Silver was believed to be phloem-mobile since it was exported from a treated leaf. Bean plants removed one-half the Ag+ from 4 liters of nutrient solution containing 50 nanomolar AgNO3 within 1.5 hours, but took 16 hours for 20 liters of solution. Images PMID:16661185

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

PubMed Central

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

1998-01-01

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

Towards a physiological basis for intensive culture of American sycamore, Platanus occidentalis L

DOE Office of Scientific and Technical Information (OSTI.GOV)

McVay, M.E.

1984-01-01

The light environment of forest canopies is complex leading to differentiation in morphological development and alteration of physiological processes that are important for the proper combination of genotype and site factors to maximize final yield. To select leaves and plants of a uniform age plastochron and leaf plastochron indices were developed for American sycamore Platanus occidentalis L. The application of these indices was useful in selecting a population of plants of uniform age and, morphological development, and useful in selecting leaves of uniform age within the zone of leaf development. Labeling studies in which leaves of specific ages were fedmore » /sup 14/CO/sub 2/ indicated that leaves of this species undergo transitions in degree and direction of export. Acropetal export of photosynthates conforms to a phyllotactic sequence, suggesting a relationship between arrangement of vascular bundles and phyllotaxy. Examination of the /sup 14/CO/sub 2/ labeling patterns in source leaves (LPI 2.0) showed rapid incorporation of label into amino acid, sugar, and organic acid chemical pools that can be used to in situ synthesis of proteins, lipids, and other structural components. However as the irradiance regime under which the plants were grown decreased, leaves partitioned more fixed /sup 14/CO/sub 2/ into starch than into the water soluble fraction from which metabolic and structural components as well as a transport species could be synthesized.« less

Evidence for metabolic activity of airborne bacteria

NASA Technical Reports Server (NTRS)

Chatigny, M. A.; Wolochow, H.

1974-01-01

Aerosols of the bacterium Serratia marcescens, and of uniformly labeled C-14 glucose were produced simultaneously and mixed in tubing leading to an aerosol chamber. During a subsequent period of about 5 hrs, carbon dioxide was produced metabolically within the chamber, and labeled material incorporated within the suspended particles first increased then decreased. This constitutes the first direct evidence of microbial metabolism of bacteria suspended in the air.

Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development[OPEN

PubMed Central

Nelson, Clark J.; Castleden, Ian

2017-01-01

We applied 15N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome. PMID:28138016

Amide or Amine: Determining the Origin of the 3300 cm−1 NH Mode in Protein SFG Spectra Using 15N Isotope Labels

PubMed Central

Weidner, Tobias; Breen, Nicholas F.; Drobny, Gary P.; Castner, David G.

2009-01-01

Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases a strong NH mode near 3300 cm−1 is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode we studied 15N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an α-helical secondary structure (LKα14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. 15N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm−1 on SiO2 and 13 cm−1 on CaF2. This clearly shows the 3300 cm−1 NH feature is associated with side chain NH stretches and not with backbone amide modes. PMID:19873996

Amide or amine: determining the origin of the 3300 cm(-1) NH mode in protein SFG spectra using 15N isotope labels.

PubMed

Weidner, Tobias; Breen, Nicholas F; Drobny, Gary P; Castner, David G

2009-11-26

Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases, a strong NH mode near 3300 cm(-1) is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain, since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode, we studied (15)N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an alpha-helical secondary structure (LKalpha14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. (15)N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm(-1) on SiO(2) and 13 cm(-1) on CaF(2). This clearly shows the 3300 cm(-1) NH feature is associated with side chain NH stretches and not with backbone amide modes.

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

PubMed Central

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

2012-01-01

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

U.S. EPA, Pesticide Product Label, PROKIL MALATHION ULV, 08/15/1991

EPA Pesticide Factsheets

2011-04-21

... to aquatic or,anl ••• In a,.a. near the aprllcatlon .It ... vl.ltln, tha tlrlt",ent Ir'l ... product In the '0 to 60 ~ •• , m.dl.n dll •• tlr (HMD) .lcron ran,., vlth no .ore than 10 ...

U.S. EPA, Pesticide Product Label, DOW 2 + 2, 02/15/1983

EPA Pesticide Factsheets

2011-04-14

... lOUIII, 1,,.1'" ""ILI, ... , 1.Ule ,p' .. , ~u, ... " tr",n tu,,'lg 01 ,"'cuth b .... Appl, at .," 1r,,·0I1,I111 ... ), ... Ov nol • .,pI1 .. h

Expression of transforming growth factor-beta1, -beta2 and -beta3 in normal and diseased canine mitral valves.

PubMed

Aupperle, H; März, I; Thielebein, J; Schoon, H-A

2008-01-01

The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.

Resolution enhancement in 13C and 15N magic-angle turning experiments with TPPM decoupling.

PubMed

McGeorge, G; Alderman, D W; Grant, D M

1999-03-01

Many solid-state spectra have been shown to have problems related to the poor proton decoupling of carbon nuclei in methylene groups under conditions of slow magic-angle turning. Two-pulse phase-modulation (TPPM) decoupling during the 2D PHORMAT chemical shift separation experiment is shown to be more effective in comparison to that obtainable at much higher spin rates using conventional CW decoupling. TPPM decoupling can also alleviate similar inadequacies when observing the 15N nucleus, particularly with NH2 groups. This is demonstrated in the 15N resonances of fully labeled l-arginine hydrochloride, where a line narrowing of about a factor of two was observed at moderate rotation rates. This significant advantage was also obtained at turning frequencies as low as 500 Hz. Copyright 1999 Academic Press.

Target-specific NMR detection of protein-ligand interactions with antibody-relayed 15N-group selective STD.

PubMed

Hetényi, Anasztázia; Hegedűs, Zsófia; Fajka-Boja, Roberta; Monostori, Éva; Kövér, Katalin E; Martinek, Tamás A

2016-12-01

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1 H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15 N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15 N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Characterization of labelling and de-labelling reagents for detection and recovery of tyrosine residue in peptide.

PubMed

Toyo'oka, Toshimasa; Mantani, Tomomi; Kato, Masaru

2003-01-01

This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Copyright 2003 John Wiley & Sons, Ltd.

[Localization of hepatocellular carcinoma with monoclonal antibodies].

PubMed

Liu, Y

1991-07-01

We prepared monoclonal antibodies (MAbs) against hepatocellular carcinoma using cell suspensions isolated from surgical fresh hepatoma specimens as antigen. Totally we got 6 strains of hybridoma cell lines stably secreting MAbs for more than 2 years. Immunocytochemically they stained positively most of the paraffin embedded hepatoma tissues (63.1 to 91.1%) without reaction to the normal liver tissues. Localization of human hepatoma with 125I or 131I labelled MAbs in nude mice was done by IV injection, which showed clear tumor image by ECT radioimmunodetection and autoradiography of tissues. The T/N ratios of different MAbs were 3.1, 3.6, 5.15 and that of HAb 18-F (ab')2 was 14.4. Among 15 patients suspected to have hepatoma and given the labelled MAb, 13 proved pathologically to be hepatocellular carcinoma.

47 CFR 15.19 - Labelling requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Labelling requirements. 15.19 Section 15.19 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL RADIO FREQUENCY DEVICES General § 15.19 Labelling requirements. (a) In addition to the requirements in part 2 of this chapter, a device subject to certification...

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

PubMed

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

2015-10-20

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

Tracing the cycling and fate of the explosive 2,4,6-trinitrotoluene in coastal marine systems with a stable isotopic tracer, 15N-[TNT

USGS Publications Warehouse

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

2015-01-01

2,4,6-Trinitrotoluene (TNT) has been used as a military explosive for over a hundred years. Contamination concerns have arisen as a result of manufacturing and use on a large scale; however, despite decades of work addressing TNT contamination in the environment, its fate in marine ecosystems is not fully resolved. Here we examine the cycling and fate of TNT in the coastal marine systems by spiking a marine mesocosm containing seawater, sediments, and macrobiota with isotopically labeled TNT (15N-[TNT]), simultaneously monitoring removal, transformation, mineralization, sorption, and biological uptake over a period of 16 days. TNT degradation was rapid, and we observed accumulation of reduced transformation products dissolved in the water column and in pore waters, sorbed to sediments and suspended particulate matter (SPM), and in the tissues of macrobiota. Bulk δ15N analysis of sediments, SPM, and tissues revealed large quantities of 15N beyond that accounted for in identifiable derivatives. TNT-derived N was also found in the dissolved inorganic N (DIN) pool. Using multivariate statistical analysis and a 15N mass balance approach, we identify the major transformation pathways of TNT, including the deamination of reduced TNT derivatives, potentially promoted by sorption to SPM and oxic surface sediments.

Optimization of identity operation in NMR spectroscopy via genetic algorithm: Application to the TEDOR experiment

NASA Astrophysics Data System (ADS)

Manu, V. S.; Veglia, Gianluigi

2016-12-01

Identity operation in the form of π pulses is widely used in NMR spectroscopy. For an isolated single spin system, a sequence of even number of π pulses performs an identity operation, leaving the spin state essentially unaltered. For multi-spin systems, trains of π pulses with appropriate phases and time delays modulate the spin Hamiltonian to perform operations such as decoupling and recoupling. However, experimental imperfections often jeopardize the outcome, leading to severe losses in sensitivity. Here, we demonstrate that a newly designed Genetic Algorithm (GA) is able to optimize a train of π pulses, resulting in a robust identity operation. As proof-of-concept, we optimized the recoupling sequence in the transferred-echo double-resonance (TEDOR) pulse sequence, a key experiment in biological magic angle spinning (MAS) solid-state NMR for measuring multiple carbon-nitrogen distances. The GA modified TEDOR (GMO-TEDOR) experiment with improved recoupling efficiency results in a net gain of sensitivity up to 28% as tested on a uniformly 13C, 15N labeled microcrystalline ubiquitin sample. The robust identity operation achieved via GA paves the way for the optimization of several other pulse sequences used for both solid- and liquid-state NMR used for decoupling, recoupling, and relaxation experiments.

10 CFR Appendix G to Subpart B of... - Uniform Test Method for Measuring the Energy Consumption of Unvented Home Heating Equipment

Code of Federal Regulations, 2014 CFR

2014-01-01

... of Unvented Home Heating Equipment G Appendix G to Subpart B of Part 430 Energy DEPARTMENT OF ENERGY..., App. G Appendix G to Subpart B of Part 430—Uniform Test Method for Measuring the Energy Consumption of... gas control value (e.g., by label) by the manufacturer. 2.4 Electrical standby mode power measurement...

Phenotypically heterogeneous deletion of the ABH antigen from the transformed bladder urothelium. A scanning electron microscope study.

PubMed

De Harven, E; He, S; Hanna, W; Bootsma, G; Connolly, J G

1987-10-01

The deletion of ABH blood group antigens from the luminal surface of the bladder mucosa in cases of well differentiated transitional cell carcinomata, and the formation of pleomorphic microvilli have both been associated with aggressive biological behaviour and invasiveness of the tumors. We have studied cold cup biopsies from 8 normal mucosae and 17 papillary transitional cell carcinomata of the urinary bladder. The aim of our study was to correlate the formation of uniform or pleomorphic microvilli with the extent of deletion of the ABH blood group antigens on the surface of normal and transformed bladder urothelium. Immunogold scanning electron microscopy (SEM) in the backscattered electron (BE) imaging mode was used for this purpose. In the normal urothelium, uniform labeling of the luminal cells was demonstrated. In well differentiated tumors, the superficial cells exhibited uniform microvilli and a heterogeneous expression of the ABH antigens, giving characteristic 'mosaic' patterns of the antigenic labeling across the mucosal surface. These patterns were sharply delimitated at cell junctions when viewed by SEM; these observations were confirmed by transmission electron microscopy. In higher grade tumors, decreased ABH antigen expression, pleomorphic microvilli and/or featureless luminal cells were observed. In the transformed urothelium, the formation of uniform microvilli appeared to precede the loss of ABH antigen in most cases.

Inconsistencies in emergency instructions on common household product labels.

PubMed

Cantrell, F Lee; Nordt, Sean Patrick; Krauss, Jamey R

2013-10-01

Human exposures to non-pharmaceutical products often results in serious injury and death annually in the United States. Studies performed more than 25 years ago described inadequate first aid advice on the majority of household products. The current study evaluates contemporary non-pharmaceutical products with respect to location, uniformity and type of their first aid and emergency contact instructions. A random, convenience sample of commercial product label information was obtained from local retail stores over an 8 month period. Twelve common non-pharmaceutical product categories, with large numbers of annual human exposures, were identified from National Poison Data Systems data. A minimum of 10 unique products for each category utilized. The following information identified: product name and manufacturer, location on container, presence and type of route-specific treatment, medical assistance referral information. A total of 259 product labels were examined. First aid/contact information was located on container: rear 162 (63 %), side 28 (11 %), front 3 (1 %), bottom 2 (0.77 %), behind label 14 (5 %), missing entirely 50 (19 %). Fifty-five products (21 %) lacked any first aid instructions. Suggested contacts for accidental poisoning: none listed 75 (29 %), physician 144 (56 %), poison control centers 102 (39 %), manufacturer 44 (17 %), "Call 911" 10 (4 %). Suggested contacts for unintentional exposure and content of first aid instructions on household products were inconsistent, frequently incomplete and at times absent. Instruction locations similarly lacked uniformity. Household product labels need to provide concise, accurate first aid and emergency contact instructions in easy-to-understand language in a universal format on product labels.

Chronic migraine headache prevention with noninvasive vagus nerve stimulation: The EVENT study.

PubMed

Silberstein, Stephen D; Calhoun, Anne H; Lipton, Richard B; Grosberg, Brian M; Cady, Roger K; Dorlas, Stefanie; Simmons, Kristy A; Mullin, Chris; Liebler, Eric J; Goadsby, Peter J; Saper, Joel R

2016-08-02

To evaluate the feasibility, safety, and tolerability of noninvasive vagus nerve stimulation (nVNS) for the prevention of chronic migraine (CM) attacks. In this first prospective, multicenter, double-blind, sham-controlled pilot study of nVNS in CM prophylaxis, adults with CM (≥15 headache d/mo) entered the baseline phase (1 month) and were subsequently randomized to nVNS or sham treatment (2 months) before receiving open-label nVNS treatment (6 months). The primary endpoints were safety and tolerability. Efficacy endpoints in the intent-to-treat population included change in the number of headache days per 28 days and acute medication use. Fifty-nine participants (mean age, 39.2 years; mean headache frequency, 21.5 d/mo) were enrolled. During the randomized phase, tolerability was similar for nVNS (n = 30) and sham treatment (n = 29). Most adverse events were mild/moderate and transient. Mean changes in the number of headache days were -1.4 (nVNS) and -0.2 (sham) (Δ = 1.2; p = 0.56). Twenty-seven participants completed the open-label phase. For the 15 completers initially assigned to nVNS, the mean change from baseline in headache days after 8 months of treatment was -7.9 (95% confidence interval -11.9 to -3.8; p < 0.01). Therapy with nVNS was well-tolerated with no safety issues. Persistent prophylactic use may reduce the number of headache days in CM; larger sham-controlled studies are needed. NCT01667250. This study provides Class II evidence that for patients with CM, nVNS is safe, is well-tolerated, and did not significantly change the number of headache days. This pilot study lacked the precision to exclude important safety issues or benefits of nVNS. © 2016 American Academy of Neurology.

Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

PubMed

Ziegel, Rebecca; Shallop, Anthony; Upadhyaya, Pramod; Jones, Roger; Tretyakova, Natalia

2004-01-20

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously methylated CG sites within the p53 tumor suppressor gene.

Impacts of water and nitrogen addition on nitrogen recovery in Haloxylon ammodendron dominated desert ecosystems.

PubMed

Cui, Xiaoqing; Yue, Ping; Gong, Yanming; Li, Kaihui; Tan, Dunyan; Goulding, Keith; Liu, Xuejun

2017-12-01

Desert ecosystems are likely to change in response to global climate change and nitrogen (N) deposition. The effects of increased precipitation and N deposition on plant growth and the N cycle largely depend on N allocation and N recovery efficiency in the plant-soil ecosystem, but there is limited research on this in desert ecosystems. Here we report results using double-labeled 15 NH 4 15 NO 3 (30 and 60kgNha -1 yr -1 ) as a tracer under ambient (no additional water addition) and enhanced precipitation (60mm water addition) in a Haloxylon ammodendron dominated ecosystem in the Gurbantunggut Desert of Northwest China. Herbaceous plants were a significantly larger sink for added 15 N than the H. ammodendron trees, and N retention varied with water and N addition, relative to growing season precipitation. The retention of added 15 N varied within the components of H. ammodendron, with the stems retaining most, followed by the assimilation branches. Soil was the dominant sink for added 15 N, in which the topsoil and subsoil respond differently to water and N addition over the two-year period. Nitrogen relative recovery percentage in the whole ecosystem ranged from 43% to 61%, lower than average recovery rate in temperate forests; N tracer recovery percentage significantly increased with water addition but decreased with enhanced N deposition. Future N cycling in central Asian deserts will depend on changes in precipitation. Copyright © 2017 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2016-10-01

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

47 CFR 15.19 - Labelling requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 1 2011-10-01 2011-10-01 false Labelling requirements. 15.19 Section 15.19 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL RADIO FREQUENCY DEVICES General § 15.19 Labelling requirements. (a) In addition to the requirements in part 2 of this chapter, a device subject to certification, or verification shall be...

Proton-Detected 15N NMR-Spectroscopy and Imaging

NASA Astrophysics Data System (ADS)

Freeman, D.; Sailasuta, N.; Sukumar, S.; Hurd, R. E.

1993-10-01

Proton detection of nitrogen-15, using gradients for coherence selection, was determined to be an effective method for obtaining spectra of 15N-labeled metabolites from extracts and biopsies of tissue infused with [15N] ammonium chloride. The advantage of gradient selection of coherence was best demonstrated by the almost complete single-shot elimination of solvent water in extracts and tissue water in biopsies. As a single-acquisition editing method in which only protons attached to 15N are detected, the potential limitations of dynamic range and motion are also reduced. Gradient-enhanced heteronuclear multiple-quantum coherence (1H[15N] HMQC) was compared with conventional HMQC, and despite selection of only one of the two heteronuclear pathways, GE-HMQC was found to be more effective for resolving the desired signal for dilute solutions; and with a single scan. In addition, effective water elimination made it possible to use the resolution advantage of a frequency-encoding dimension in proton-detected 15N imaging experiments. The limit of detection of the method at 500 MHz was 0.7 mM in 16 scans from a total volume of 400 μl. Signals from tissue extracts were observable in less than one minute for kidney, heart, brain, and muscle. Proton-detected 15N GE-HMQC images with a voxel size of 39 × 78 × 625 μm were obtained at 600 MHz from a 4 mM (1.6 μmol) 15N urea sample in less than four hours. Distribution of [15N] urea in the kidney was observed in a 600 MHz GE-HMQC image of the papilla and some cortical structures.

Ferritin conjugates as specific magnetic labels. Implications for cell separation.

PubMed Central

Odette, L L; McCloskey, M A; Young, S H

1984-01-01

Concanavalin A coupled to the naturally occurring iron storage protein ferritin is used to label rat erythrocytes and increase the cells' magnetic susceptibility. Labeled cells are introduced into a chamber containing spherical iron particles and the chamber is placed in a uniform 5.2 kG (gauss) magnetic field. The trajectory of cells in the inhomogeneous magnetic field around the iron particles and the polar distributions of cells bound to the iron particles compare well with the theoretical predictions for high gradient magnetic systems. On the basis of these findings we suggest that ferritin conjugated ligands can be used for selective magnetic separation of labeled cells. Images FIGURE 2 PMID:6743752

View the label before you view the movie: A field experiment into the impact of Portion size and Guideline Daily Amounts labelling on soft drinks in cinemas

PubMed Central

2011-01-01

Background Large soft drink sizes increase consumption, and thereby contribute to obesity. Portion size labelling may help consumers to select more appropriate food portions. This study aimed to assess the effectiveness of portion size and caloric Guidelines for Daily Amounts (GDA) labelling on consumers' portion size choices and consumption of regular soft drinks. Methods A field experiment that took place on two subsequent evenings in a Dutch cinema. Participants (n = 101) were asked to select one of five different portion sizes of a soft drink. Consumers were provided with either portion size and caloric GDA labelling (experimental condition) or with millilitre information (control condition). Results Labelling neither stimulated participants to choose small portion sizes (OR = .75, p = .61, CI: .25 - 2.25), nor did labelling dissuade participants to choose large portion sizes (OR = .51, p = .36, CI: .12 - 2.15). Conclusions Portion size and caloric GDA labelling were found to have no effect on soft drink intake. Further research among a larger group of participants combined with pricing strategies is required. The results of this study are relevant for the current public health debate on food labelling. PMID:21645373

Utilization of organic nitrogen by arbuscular mycorrhizal fungi-is there a specific role for protists and ammonia oxidizers?

PubMed

Bukovská, Petra; Bonkowski, Michael; Konvalinková, Tereza; Beskid, Olena; Hujslová, Martina; Püschel, David; Řezáčová, Veronika; Gutiérrez-Núñez, María Semiramis; Gryndler, Milan; Jansa, Jan

2018-04-01

Arbuscular mycorrhizal (AM) fungi can significantly contribute to plant nitrogen (N) uptake from complex organic sources, most likely in concert with activity of soil saprotrophs and other microbes releasing and transforming the N bound in organic forms. Here, we tested whether AM fungus (Rhizophagus irregularis) extraradical hyphal networks showed any preferences towards certain forms of organic N (chitin of fungal or crustacean origin, DNA, clover biomass, or albumin) administered in spatially discrete patches, and how the presence of AM fungal hyphae affected other microbes. By direct 15 N labeling, we also quantified the flux of N to the plants (Andropogon gerardii) through the AM fungal hyphae from fungal chitin and from clover biomass. The AM fungal hyphae colonized patches supplemented with organic N sources significantly more than those receiving only mineral nutrients, organic carbon in form of cellulose, or nothing. Mycorrhizal plants grew 6.4-fold larger and accumulated, on average, 20.3-fold more 15 N originating from the labeled organic sources than their nonmycorrhizal counterparts. Whereas the abundance of microbes (bacteria, fungi, or Acanthamoeba sp.) in the different patches was primarily driven by patch quality, we noted a consistent suppression of the microbial abundances by the presence of AM fungal hyphae. This suppression was particularly strong for ammonia oxidizing bacteria. Our results indicate that AM fungi successfully competed with the other microbes for free ammonium ions and suggest an important role for the notoriously understudied soil protists to play in recycling organic N from soil to plants via AM fungal hyphae.

Rigid, Perfectly Plastic Analysis of Ring-Stiffened Shells Under Dynamic Loading

DTIC Science & Technology

1986-08-01

Elastic-Viscoplastic Response of Clamped Beams Under Uniformly Distributed Impulse, Technical Report AFML-TR-68-396, Jan 1969. 15. Save , M. A. and...Edition, 1959, pp. 466-471 (Chapter 15). A-4. Save , M. A. and Massonnett, C. E., Plastic Analysis and Design of Plates, Shells, and Discs, North-Holland...TYPE TYESHORT (2 ɞ) HIGH LOAnD p > I + CONDITIONS u C 2 (-1) 0 T 1 0 x < MOMENT m Ix, T) 2 +1 OR -RESULTANT Lxo u.=-g- MEMBRANE n,= RESULTANT

Structural analysis of N-glycans by the glycan-labeling method using 3-aminoquinoline-based liquid matrix in negative-ion MALDI-MS.

PubMed

Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-ichirou; Tanaka, Koichi

2012-11-06

Negative-ion fragmentation of underivatized N-glycans has been proven to be more informative than positive-ion fragmentation. Fluorescent labeling via reductive amination is often employed for glycan analysis, but little is known about the influence of the labeling group on negative-ion fragmentation. We previously demonstrated that the on-target glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) liquid matrix enables highly sensitive, rapid, and quantitative N-glycan profiling analysis. The current study investigates the suitability of 3AQ-labeled N-glycans for structural analysis based on negative-ion collision-induced dissociation (CID) spectra. 3AQ-labeled N-glycans exhibited simple and informative CID spectra similar to those of underivatized N-glycans, with product ions due to cross-ring cleavages of the chitobiose core and ions specific to two antennae (D and E ions). The interpretation of diagnostic fragment ions suggested for underivatized N-glycans could be directly applied to the 3AQ-labeled N-glycans. However, fluorescently labeled N-glycans by conventional reductive amination, such as 2-aminobenzamide (2AB)- and 2-pyrydilamine (2PA)-labeled N-glycans, exhibited complicated CID spectra consisting of numerous signals formed by dehydration and multiple cleavages. The complicated spectra of 2AB- and 2PA-labeled N-glycans was found to be due to their open reducing-terminal N-acetylglucosamine (GlcNAc) ring, rather than structural differences in the labeling group in the N-glycan derivative. Finally, as an example, the on-target 3AQ labeling method followed by negative-ion CID was applied to structurally analyze neutral N-glycans released from human epidermal growth factor receptor type 2 (HER2) protein. The glycan-labeling method using 3AQ-based liquid matrix should facilitate highly sensitive quantitative and qualitative analyses of glycans.

Fee-Based Versus Fixed Income Reimbursement: A Comparative Analysis of Staff Physicians and Civilian Health and Medical Program of the Uniformed Services (CHAMPUS) Partnership Physicians at Blanchfield Army Community Hospital, Fort Campbell, Kentucky.

DTIC Science & Technology

1992-07-01

vaginitis and vulvovaginitis , appeared a total of 117 times in the data set. While 616.1 was overall ranked as the tenth most frequently used ICD-9...124) 9. 462 Acute Pharyngitis (n-118) 10. 616.1 Vaginitis and Vulvovaginitis (n-117) 11. 692.9 Contact Dermatitis and other Eczema, Unspecified Cause...Urethra and Urinary Tract Comparative Analy3is 60 (n-17) 33. 464.1 Acute Tracheitis (n-16) 34. 616.1 Vaginitis and Vulvovaginitis (n-15) 35. 910.5

76 FR 52441 - Summary of Benefits and Coverage and the Uniform Glossary

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-22

... facts label that includes examples to illustrate common benefits scenarios (including pregnancy and... plan or coverage for common benefits scenarios, including pregnancy and serious or chronic medical...

[Effects of clipping on nitrogen allocation strategy and compensatory growth of Leymus chinensis under saline-alkali conditions].

PubMed

Zheng, Cong Cong; Wang, Yong Jing; Sun, Hao; Wang, Xin Yu; Gao, Ying Zhi

2017-07-18

Soil salinization and overgrazing are two main factors limiting animal husbandry in the Songnen Grassland. Leymus chinensis is a dominant rhizome grass, resistant to grazing as well as to-lerant to salt stress. Foliar labeled with 15 N-urea was used to study the nitrogen allocation strategy and compensatory growth response to clipping under saline-alkali conditions. The results showed that the total absorbed 15 N allocated to the aboveground part was more than 60%. Compared with the control treatment (no saline-alkali, no clipping), saline-alkali increased the distribution of 15 N by 5.1% in root; the 15 N distribution into aboveground in the moderate clipping and saline-alkali treatment was 11.6% higher than that of the control, exhibiting over-compensatory growth of aboveground biomass and total biomass, however, 15 N allocated to stem base was significantly increased by 9.5% under severe clipping level and saline-alkali addition, showing under-compensatory growth of shoot, root and total biomass. These results suggested that L. chinensis adapted to mode-rate clipping by over-compensatory growth under salt-alkali stress condition. However, L. chinensis would take a relatively conservative growth strategy through the enhanced N allocation to stem base for storage under severe saline-alkali and clipping conditions.

Assimilate partitioning during reproductive growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Finazzo, S.F.; Davenport, T.L.

1987-04-01

Leaves having various phyllotactic relationships to fruitlets were labeled for 1 hour with 10/sub r/Ci of /sup 14/CO/sub 2/. Fruitlets were also labeled. Fruitlets did fix /sup 14/CO/sub 2/. Translocation of radioactivity from the peel into the fruit occurred slowly and to a limited extent. No evidence of translocation out of the fruitlets was observed. Assimilate partitioning in avocado was strongly influenced by phyllotaxy. If a fruit and the labeled leaf had the same phyllotaxy then greater than 95% of the radiolabel was present in this fruit. When the fruit did not have the same phyllotaxy as the labeled leaf,more » the radiolabel distribution was skewed with 70% of the label going to a single adjacent position. Avocado fruitlets exhibit uniform labeling throughout a particular tissue. In avocado, assimilates preferentially move from leaves to fruits with the same phyllotaxy.« less

75 FR 12470 - Automotive Fuel Ratings, Certification and Posting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-16

... the Secretary, Room H-135 (Annex M), 600 Pennsylvania Avenue, N.W., Washington, D.C. 20580, in the... with the Petroleum Marketing Practices Act (``PMPA''), 15 U.S.C. 2801 et seq.\\3\\ The Rule originally... the specific labeling requirements for biodiesel fuels required by Section 205 of EISA, 42 U.S.C...

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

USDA-ARS?s Scientific Manuscript database

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

U.S. EPA, Pesticide Product Label, HENRY FIELD'S NEW IMPROVED BUG DUST, 04/17/1975

EPA Pesticide Factsheets

2011-04-14

... 'Qu.:utiy UIIJt.'f sev~re disease conditj<)II~. ... titan to for'm) V<.i\\~I!Y I.ili

A novel 15N tracer approach for the quantification of N2 and N2O emissions from soil incubations in a completely automated laboratory set up

NASA Astrophysics Data System (ADS)

Scheer, Clemens; Dannenmann, Michael; Meier, Rudolf

2015-04-01

The microbial mediated production of nitrous oxide (N2O) and its reduction to dinitrogen (N2) via denitrification represents a loss of nitrogen (N) from fertilised agro-ecosystems to the atmosphere. Although denitrification has received great interest by biogeochemists in the last decades, the magnitude of N2lossesand related N2:N2O ratios from soils still are largely unknown due to methodical constraints. We present a novel 15N tracer approach, based on a previous developed tracer method to study denitrification in pure bacterial cultures which was modified for the use on soil incubations in a completely automated laboratory set up. The method uses a background air in the incubation vessels that is replaced with a helium-oxygen gas mixture with a 50-fold reduced N2 background (2 % v/v). This method allows for a direct and sensitive quantification of the N2 and N2O emissions from the soil with isotope-ratio mass spectrometry after 15N labelling of denitrification N substrates and minimises the sensitivity to the intrusion of atmospheric N2 at the same time. The incubation set up was used to determine the influence of different soil moisture levels on N2 and N2O emissions from a sub-tropical pasture soil in Queensland/Australia. The soil was labelled with an equivalent of 50 μg-N per gram dry soil by broadcast application of KNO3solution (4 at.% 15N) and incubated for 3 days at 80% and 100% water filled pore space (WFPS), respectively. The headspace of the incubation vessel was sampled automatically over 12hrs each day and 3 samples (0, 6, and 12 hrs after incubation start) of headspace gas analysed for N2 and N2O with an isotope-ratio mass spectrometer (DELTA V Plus, Thermo Fisher Scientific, Bremen, Germany(. In addition, the soil was analysed for 15N NO3- and NH4+ using the 15N diffusion method, which enabled us to obtain a complete N balance. The method proved to be highly sensitive for N2 and N2O emissions detecting N2O emissions ranging from 20 to 627 μN kg-1soil-1hr-1and N2 emissions ranging from 4.2 to 43 μN kg-1soil-1hr-1for the different treatments. The main end-product of denitrification was N2O for both water contents with N2 accounting for 9% and 13% of the total denitrification losses at 80% and 100%WFPS, respectively. Between 95-100% of the added 15N fertiliser could be recovered. Gross nitrification over the 3 days amounted to 8.6 μN g-1 soil-1 and 4.7 μN g-1 soil-1, denitrification to 4.1 μN g-1 soil-1 and 11.8 μN g-1 soil-1at 80% and 100%WFPS, respectively. The results confirm that the tested method allows for a direct and highly sensitive detection of N2 and N2O fluxes from soils and hence offers a sensitive tool to study denitrification and N turnover in terrestrial agro-ecosystems.

Determination of ¹⁵N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

PubMed

Ullmann-Zeunert, Lynn; Muck, Alexander; Wielsch, Natalie; Hufsky, Franziska; Stanton, Mariana A; Bartram, Stefan; Böcker, Sebastian; Baldwin, Ian T; Groten, Karin; Svatoš, Aleš

2012-10-05

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Protein-bound D-amino acids, and to a lesser extent lysinoalanine, decrease true ileal protein digestibility in minipigs as determined with (15)N-labeling.

PubMed

de Vrese, M; Frik, R; Roos, N; Hagemeister, H

2000-08-01

Heat and alkali treatment of food may increase the concentrations of protein-bound D-amino acids and cross-links such as lysinoalanine (LAL). To examine how protein treatment affects digestibility, purified test meals [total protein 150 g/kg dry matter (DM), 0.44 MJ/(kg BW(0.75). d)] were prepared, containing (g/kg DM) casein, 75; beta-lactoglobulin, 50; or wheat protein, 40. Each was (15)N-labeled. Test proteins were used either in their native form or after treatment for 6 or 24 h at 65 degrees C, pH 10.5-11.5. Each meal was fed to nine adult miniature pigs (twofold complete cross-classification). Chyme was collected continuously over 33 h postprandially via T-fistulas in the terminal ileum, and digestibilities of test proteins and individual L- and D-amino acids were calculated on the basis of recovery of (15)N and the respective amino acids in the chyme. Treatment of casein, beta-lactoglobulin or wheat protein for 24 h increased levels of D-amino acid residues. L-Asparagine and aspartate (L-Asx) were particularly susceptible; 14. 7 +/- 0.4, 11.7 +/- 0.2 and 11.0 +/- 0.9%, respectively, underwent racemization. LAL levels increased in parallel; 11.7 +/- 0.3, 13.6 +/- 0 and 14.8 +/- 0.0%, respectively, of total lysine was converted to LAL. At the same time, prececal protein digestibility was decreased by 13.4 +/- 2.3, 15.3 +/- 1.4 and 17.8 +/- 1.2% units, respectively (P < 0.05; mean +/- SEM, n = 9). Digestibility of individual L-amino acids decreased by 10-15%, but L-amino acids prone to peptic cleavage, such as L-phenylalanine and L-tyrosine, were not affected. Digestibilities of D-amino acids and LAL were approximately 35%. It seems that mainly D-amino acids, and to a lesser extent LAL, were responsible for lower digestibility by interfering with peptic cleavage.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan Lin; Liu Fuyi; Armentrout, P.B.

The kinetic energy dependences of the reactions of Fe{sub n}{sup +} (n=1-19) with N{sub 2} are studied in a guided ion beam tandem mass spectrometer over the energy range of 0-15 eV. In addition to collision-induced dissociation forming Fe{sub m}{sup +} ions, which dominate the product spectra, a variety of Fe{sub m}N{sub 2}{sup +} and Fe{sub m}N{sup +} product ions, where m{<=}n, is observed. All processes are observed to exhibit thresholds. Fe{sub m}{sup +}-N and Fe{sub m}{sup +}-2N bond energies as a function of cluster size are derived from the threshold analysis of the kinetic energy dependences of the endothermicmore » reactions. The trends in this thermochemistry are compared to the isoelectronic D{sub 0}(Fe{sub n}{sup +}-CH), and to bulk phase values. A fairly uniform barrier of 0.48{+-}0.03 eV at 0 K is observed for formation of the Fe{sub n}N{sub 2}{sup +} product ions (n=12, 15-19) and can be related to the rate-limiting step in the Haber process for catalytic ammonia production.« less

Effects of a Voluntary Front-of-Pack Nutrition Labelling System on Packaged Food Reformulation: The Health Star Rating System in New Zealand

PubMed Central

Eyles, Helen; Choi, Yeun-Hyang

2017-01-01

Interpretive, front-of-pack (FOP) nutrition labels may encourage reformulation of packaged foods. We aimed to evaluate the effects of the Health Star Rating (HSR), a new voluntary interpretive FOP labelling system, on food reformulation in New Zealand. Annual surveys of packaged food and beverage labelling and composition were undertaken in supermarkets before and after adoption of HSR i.e., 2014 to 2016. Outcomes assessed were HSR uptake by food group star ratings of products displaying a HSR label; nutritional composition of products displaying HSR compared with non-HSR products; and the composition of products displaying HSR labels in 2016 compared with their composition prior to introduction of HSR. In 2016, two years after adoption of the voluntary system, 5.3% of packaged food and beverage products surveyed (n = 807/15,357) displayed HSR labels. The highest rates of uptake were for cereals, convenience foods, packaged fruit and vegetables, sauces and spreads, and ‘Other’ products (predominantly breakfast beverages). Products displaying HSR labels had higher energy density but had significantly lower mean saturated fat, total sugar and sodium, and higher fibre, contents than non-HSR products (all p-values < 0.001). Small but statistically significant changes were observed in mean energy density (−29 KJ/100 g, p = 0.002), sodium (−49 mg/100 g, p = 0.03) and fibre (+0.5 g/100 g, p = 0.001) contents of HSR-labelled products compared with their composition prior to adoption of HSR. Reformulation of HSR-labelled products was greater than that of non-HSR-labelled products over the same period, e.g., energy reduction in HSR products was greater than in non-HSR products (−1.5% versus −0.4%), and sodium content of HSR products decreased by 4.6% while that of non-HSR products increased by 3.1%. We conclude that roll-out of the voluntary HSR labelling system is driving healthier reformulation of some products. Greater uptake across the full food supply should improve population diets. PMID:28829380

Effects of a Voluntary Front-of-Pack Nutrition Labelling System on Packaged Food Reformulation: The Health Star Rating System in New Zealand.

PubMed

Mhurchu, Cliona Ni; Eyles, Helen; Choi, Yeun-Hyang

2017-08-22

Interpretive, front-of-pack (FOP) nutrition labels may encourage reformulation of packaged foods. We aimed to evaluate the effects of the Health Star Rating (HSR), a new voluntary interpretive FOP labelling system, on food reformulation in New Zealand. Annual surveys of packaged food and beverage labelling and composition were undertaken in supermarkets before and after adoption of HSR i.e., 2014 to 2016. Outcomes assessed were HSR uptake by food group star ratings of products displaying a HSR label; nutritional composition of products displaying HSR compared with non-HSR products; and the composition of products displaying HSR labels in 2016 compared with their composition prior to introduction of HSR. In 2016, two years after adoption of the voluntary system, 5.3% of packaged food and beverage products surveyed ( n = 807/15,357) displayed HSR labels. The highest rates of uptake were for cereals, convenience foods, packaged fruit and vegetables, sauces and spreads, and 'Other' products (predominantly breakfast beverages). Products displaying HSR labels had higher energy density but had significantly lower mean saturated fat, total sugar and sodium, and higher fibre, contents than non-HSR products (all p -values < 0.001). Small but statistically significant changes were observed in mean energy density (-29 KJ/100 g, p = 0.002), sodium (-49 mg/100 g, p = 0.03) and fibre (+0.5 g/100 g, p = 0.001) contents of HSR-labelled products compared with their composition prior to adoption of HSR. Reformulation of HSR-labelled products was greater than that of non-HSR-labelled products over the same period, e.g., energy reduction in HSR products was greater than in non-HSR products (-1.5% versus -0.4%), and sodium content of HSR products decreased by 4.6% while that of non-HSR products increased by 3.1%. We conclude that roll-out of the voluntary HSR labelling system is driving healthier reformulation of some products. Greater uptake across the full food supply should improve population diets.

Hunter AAF, Savannah, Georgia. Revised Uniform Summary of Surface Weather Observations (RUSSWO)

DTIC Science & Technology

1970-11-18

Center of AIT/I.1Q-11 No Chile 13 Ft. to F~po 62 Rnwy 6 mar 62 Located 471 pt. 883 of Ros. No Chgc No Chile 15 Ft. to Feb 67 7 Mar 67 1. Located 300 Ft...N of lbwy 09 No Chile No Chile 25 Ft. o Feb 68 at 500 Ft Marker. 2. Located 300 Ft. N of Rawy 27 No Chile 15 Ft. at 500 Ft. Marker. 8 Mar 68 1...Located 500 Ft.from. Center Linc No Chile No Chile 20 Ft. May 70 and 1500 Pt from end of 1"w 95 2. Located 1100 Ft from Center Lir No Chge J-- 16 Ft and

Buckley ANGB, Aurora, Colorado. Revised Uniform Summary of Surface Weather Observations (RUSSWO). Parts A-F

DTIC Science & Technology

1981-02-18

OSURATIKM) 23036 bUCALEY ANG8 Qb-7.37 SYATIALL WATHER ’. 2100-2300 6LASO Nouni IINYS15) 1.3 446 ?-to 11.16 17-1 21 .3V 3.33 34.46 41.0 4.33t 1116 WOW N . 19...bulb 3398619 561 6 0ol 2,63? 930 ,. 1 1 t,% 193720m~nK( �.44.9 810 3 I I z ~ -- - -. - - I I A U GSAFETAC PSYCHROMETRIC SUMMARY AIR WATHER

Critical zone properties control the fate of nitrogen during experimental rainfall in montane forests of the Colorado Front Range

USGS Publications Warehouse

Hinckley, Eve-Lyn S.; Ebel, Brian A.; Barnes, Rebecca T.; Murphy, Sheila F.; Anderson, Suzanne P.

2017-01-01

Several decades of research in alpine ecosystems have demonstrated links among the critical zone, hydrologic response, and the fate of elevated atmospheric nitrogen (N) deposition. Less research has occurred in mid-elevation forests, which may be important for retaining atmospheric N deposition. To explore the fate of N in the montane zone, we conducted plot-scale experimental rainfall events across a north–south transect within a catchment of the Boulder Creek Critical Zone Observatory. Rainfall events mimicked relatively common storms (20–50% annual exceedance probability) and were labeled with 15N-nitrate (NO3−">NO−3NO3−) and lithium bromide tracers. For 4 weeks, we measured soil–water and leachate concentrations of Br−, 15NO3−,">15NO−3,15NO3−, and NO3−">NO−3NO3− daily, followed by recoveries of 15N species in bulk soils and microbial biomass. Tracers moved immediately into the subsurface of north-facing slope plots, exhibiting breakthrough at 10 and 30 cm over 22 days. Conversely, little transport of Br− or 15NO3−">15NO−315NO3− occurred in south-facing slope plots; tracers remained in soil or were lost via pathways not measured. Hillslope position was a significant determinant of soil 15N-NO3−">NO−3NO3− recoveries, while soil depth and time were significant determinants of 15N recovery in microbial biomass. Overall, 15N recovery in microbial biomass and leachate was greater in upper north-facing slope plots than lower north-facing (toeslope) and both south-facing slope plots in August; by October, 15N recovery in microbial N biomass within south-facing slope plots had increased substantially. Our results point to the importance of soil properties in controlling the fate of N in mid-elevation forests during the summer season.

Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles

PubMed Central

Neumoin, Alexey; Cohen, Leah S.; Arshava, Boris; Tantry, Subramanyam; Becker, Jeffrey M.; Zerbe, Oliver; Naider, Fred

2009-01-01

Abstract The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for α-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [15N], [15N, 13C], [15N, 13C, 2H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three α-helices encompassing residues 39–47, 49–72, and 80–103, with higher flexibility around the internal Arg58 site of TM1. RMSD values of individually superimposed helical segments 39–47, 49–72, and 80–103 were 0.25 ± 0.10 Å, 0.40 ± 0.13 Å, and 0.57 ± 0.19 Å, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G56VRSG60 region. 15N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor. PMID:19383463

Spontaneous 15N Nuclear Spin Hyperpolarization in Metal-Free Activation of Parahydrogen by Molecular Tweezers

PubMed Central

2018-01-01

The ability of frustrated Lewis pairs (FLPs) to activate H2 is of significant interest for metal-free catalysis. The activation of H2 is also the key element of parahydrogen-induced polarization (PHIP), one of the nuclear spin hyperpolarization techniques. It is demonstrated that o-phenylene-based ansa-aminoboranes (AABs) can produce 1H nuclear spin hyperpolarization through a reversible interaction with parahydrogen at ambient temperatures. Heteronuclei are useful in NMR and MRI as well because they have a broad chemical shift range and long relaxation times and may act as background-free labels. We report spontaneous formation of 15N hyperpolarization of the N–H site for a family of AABs. The process is efficient at the high magnetic field of an NMR magnet (7 T), and it provides up to 350-fold 15N signal enhancements. Different hyperpolarization effects are observed with various AAB structures and in a broad temperature range. Spontaneous hyperpolarization, albeit an order of magnitude weaker than that for 15N, was also observed for 11B nuclei. PMID:29401399

Rapid analysis of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed-phase ultra-performance liquid chromatography with fluorescence detection and electrospray ionization time-of-flight mass spectrometry.

PubMed

Kurihara, Takamasa; Min, Jun Zhe; Hirata, Asuka; Toyo'oka, Toshimasa; Inagaki, Shinsuke

2009-05-01

Rapid, selective and sensitive determination of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The asparaginyl-oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel-permeation chromatography were labeled with 1-pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF-MS. The PSC-labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI-TOF-MS. As the results, 15, eight and four kinds of N-linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N-linked oligosaccharides in various glycoproteins seems to be possible.

Manure incorporation reduces environmental nitrogen loss while sustaining crop productivity in the subtropical wheat-maize rotation system: A comprehensive study of nitrogen cycling and balance

NASA Astrophysics Data System (ADS)

Zhou, Minghua; Zhu, Bo; Butterbach-Bahl, klaus; Brüggemann, Nicolas

2016-04-01

Balancing nitrogen (N) budgets of agricultural systems is essential for sustaining yields at lower environmental costs. The knowledge, however, of total N budgets of agricultural systems including all N fluxes is still rare in the literature. Here, we applied a combination of monitoring in situ N fluxes and field 15N tracer and 15N isotope dilution techniques to investigate the effects of different N fertilizers (control, synthetic fertilizer, 60% synthetic fertilizer N plus 40% pig manure N, pig manure only applied at the same N rate 280 kg N ha-1 yr-1) on N pools, cycling processes, fluxes and total N balances in a subtropical wheat-maize rotation system of China. Nitrate leaching and NH3 volatilization were main hydrological and gaseous N loss pathways, respectively. The warm and wet maize season was associated with significantly larger environmental N losses than the cooler and drier wheat season. The field 15N tracing experiment showed that the wheat system had high N retention capacity (˜50% of 15N application) but with short residence time. I.e. 90% of soil residual 15N labelled fertilizer in the wheat system were utilized by plants or lost to the environment in the subsequent maize season. Our annual total N balances of the different treatments revealed that combined synthetic and organic fertilization or manure only maintained the same level of yields and led to significantly lower N losses and higher N retention, even though larger NH3 volatilization losses were caused by manure incorporation. Thus, our study suggests that a combination of synthetic and organic N fertilizers is suitable for sustaining agricultural productivity while reducing environmental N losses through fostering interactions between the soil C and N cycle.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

PubMed

Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-05-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. © 2016 by the Society for the Study of Reproduction, Inc.

Stable isotope-assisted NMR characterization of interaction between lipid A and sarcotoxin IA, a cecropin-type antibacterial peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yagi-Utsumi, Maho; Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603; Yamaguchi, Yoshiki

Highlights: ► Recombinant sarcotoxin IA was successfully produced with {sup 13}C- and {sup 15}N-labeling. ► Sarcotoxin IA adopts an N-terminal α-helix upon binding to lipid A-embedding micelles. ► Two lysine residues are involved in lipid A-mediated antibacterial activities. -- Abstract: Sarcotoxin IA is a 39-residue cecropin-type peptide from Sarcophaga peregrina. This peptide exhibits antibacterial activity against Gram-negative bacteria through its interaction with lipid A, a core component of lipopolysaccharides. To acquire detailed structural information on this specific interaction, we performed NMR analysis using bacterially expressed sarcotoxin IA analogs with {sup 13}C- and {sup 15}N-labeling along with lipid A-embedding micelles composedmore » of dodecylphosphocholine. By inspecting the stable isotope-assisted NMR data, we revealed that the N-terminal segment (Leu3–Arg18) of sarcotoxin IA formed an amphiphilic α-helix upon its interaction with the aqueous micelles. Furthermore, chemical shift perturbation data indicated that the amino acid residues displayed on this α-helix were involved in the specific interaction with lipid A. On the basis of these data, we successfully identified Lys4 and Lys5 as key residues in the interaction with lipid A and the consequent antibacterial activity. Therefore, these results provide unique information for designing chemotherapeutics based on antibacterial peptide structures.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loreto, F.; Ciccioli, P.; Cecinato, A.

1996-04-01

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

Limit of detection of 15{sub N} by gas-chromatography atomic emission detection: Optimization using an experimental design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deruaz, D.; Bannier, A.; Pionchon, C.

1995-08-01

This paper deals with the optimal conditions for the detection of {sup 15}N determined using a four-factor experimental design from [2{sup 13}C,-1,3 {sup 15}N] caffeine measured with an atomic emission detector (AED) coupled to gas chromatography (GC). Owing to the capability of a photodiodes array, AED can simultaneously detect several elements using their specific emission lines within a wavelength range of 50 nm. So, the emissions of {sup 15}N and {sup 14}N are simultaneously detected at 420.17 nm and 421.46 nm respectively. Four independent experimental factors were tested (1) helium flow rate (plasma gas); (2) methane pressure (reactant gas); (3)more » oxygen pressure; (4) hydrogen pressure. It has been shown that these four gases had a significant influence on the analytical response of {sup 15}N. The linearity of the detection was determined using {sup 15}N amounts ranging from 1.52 pg to 19 ng under the optimal conditions obtained from the experimental design. The limit of detection was studied using different methods. The limits of detection of {sup 15}N was 1.9 pg/s according to the IUPAC method (International-Union of Pure and Applied Chemistry). The method proposed by Quimby and Sullivan gave a value of 2.3 pg/s and that of Oppenheimer gave a limit of 29 pg/s. For each determination, and internal standard: 1-isobutyl-3.7 dimethylxanthine was used. The results clearly demonstrate that GC AED is sensitive and selective enough to detect and measure {sup 15}N-labelled molecules after gas chromatographic separation.« less

Effects of elevated CO2 on soil organic matter turnover and plant nitrogen uptake: First results from a dual labeling mesocosm experiment

NASA Astrophysics Data System (ADS)

Eder, Lucia Muriel; Weber, Enrico; Schrumpf, Marion; Zaehle, Sönke

2017-04-01

The response of plant growth to elevated concentrations of CO2 (eCO2) is often constrained by plant nitrogen (N) uptake. To overcome potential N limitation, plants may invest photosynthetically fixed carbon (C) into N acquiring strategies, including fine root biomass, root exudation, or C allocation to mycorrhizal fungi. In turn, these strategies may affect the decomposition of soil organic matter, leading to uncertainties in net effects of eCO2 on C storage. To gain more insight into these plant-soil C-N-interactions, we combined C and N stable isotope labeling in a mesocosm experiment. Saplings of Fagus sylvatica L. were exposed to a 13CO2 enriched atmosphere at near ambient (380 ppm) or elevated (550 ppm) CO2 concentrations for four months of the vegetation period in 2016. Aboveground and belowground net CO2 fluxes were measured separately and the 13C label enabled partitioning of total soil CO2 efflux into old, soil derived and new, plant-derived C. We used ingrowth cores to assess effects of eCO2on belowground C allocation and plant N uptake in more detail and in particular we evaluated the relative importance of ectomycorrhizal associations. In the soil of each sapling, ingrowth cores with different mesh sizes allowed fine roots or only mycorrhizal hyphae to penetrate. In one type of ingrowth core each, we incorporated fine root litter that was enriched in 15N. Additionally, total N uptake was estimated by using 15N enriched saplings and unlabeled control plants. We found that eCO2 increased aboveground net CO2 exchange rates by 19% and total soil respiration by 11%. The eCO2 effect for GPP and also for NPP was positive (+23% and +11%, respectively). By combining gaseous C fluxes with data on new and old C stocks in bulk soil and plants through destructive harvesting in late autumn 2016, we will be able to infer net effects of eCO2 on the fate of C in these mesocosms. Biomass allocation patterns can reveal physiological responses to high C availability under potentially constrained N availability. Together with data on biomass production within the ingrowth cores these results elucidate mechanisms affecting soil C storage and plant N uptake under eCO2.

Evaluation of Antiatherogenic Properties of Ezetimibe Using 3H-Labeled Low-Density-Lipoprotein Cholesterol and 99mTc-cAbVCAM1-5 SPECT in ApoE-/- Mice Fed the Paigen Diet.

PubMed

Dumas, Laurent S; Briand, François; Clerc, Romain; Brousseau, Emmanuel; Montemagno, Christopher; Ahmadi, Mitra; Bacot, Sandrine; Soubies, Audrey; Perret, Pascale; Riou, Laurent M; Devoogdt, Nick; Lahoutte, Tony; Barone-Rochette, Gilles; Fagret, Daniel; Ghezzi, Catherine; Sulpice, Thierry; Broisat, Alexis

2017-07-01

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E -/- mice. Methods: Apolipoprotein E -/- mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice ( n = 15), we intravenously injected 3 H-cholesteryl oleate-labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set ( n = 20), we used the imaging agent 99m Tc-cAbVCAM1-5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set ( n = 21), we compared VCAM-1 expression with 99m Tc-cAbVCAM1-5 uptake in various tissues. Results: Mice treated with ezetimibe showed a 173% higher LDL-cholesteryl ester plasma disappearance rate ( P < 0.001 vs. control) after 3 H-cholesteryl oleate-labeled LDL injection. At 96 h after injection, the hepatic fraction of 3 H-tracer was 61% lower in mice treated with ezetimibe ( P < 0.001). Meanwhile, LDL-derived 3 H-cholesterol excretion in the feces was 107% higher ( P < 0.001). The antiatherogenic effect of ezetimibe monitored by 99m Tc-cAbVCAM1-5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; P < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with 99m Tc-cAbVCAM1-5 uptake ( r = 0.75; P < 0.05). Conclusion: Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry

NASA Astrophysics Data System (ADS)

Domratcheva, Tatiana; Hartmann, Elisabeth; Schlichting, Ilme; Kottke, Tilman

2016-03-01

BLUF (blue light sensor using flavin) domains regulate the activity of various enzymatic effector domains in bacteria and euglenids. BLUF features a unique photoactivation through restructuring of the hydrogen-bonding network as opposed to a redox reaction or an isomerization of the chromophore. A conserved glutamine residue close to the flavin chromophore plays a central role in the light response, but the underlying modification is still unclear. We labelled this glutamine with 15N in two representative BLUF domains and performed time-resolved infrared double difference spectroscopy. The assignment of the signals was conducted by extensive quantum chemical calculations on large models with 187 atoms reproducing the UV-vis and infrared signatures of BLUF photoactivation. In the dark state, the comparatively low frequency of 1,667 cm-1 is assigned to the glutamine C=O accepting a hydrogen bond from tyrosine. In the light state, the signature of a tautomerised glutamine was extracted with the C=N stretch at ~1,691 cm-1 exhibiting the characteristic strong downshift by 15N labelling. Moreover, an indirect isotope effect on the flavin C4=O stretch was found. We conclude that photoactivation of the BLUF receptor does not only involve a rearrangement of hydrogen bonds but includes a change in covalent bonds of the protein.

Radiolabeling and in vitro and in vivo characterization of [18F]FB-[R(8,15,21), L17]-VIP as a PET imaging agent for tumor overexpressed VIP receptors.

PubMed

Cheng, Dengfeng; Yin, Duanzhi; Li, Gucai; Wang, Mingwei; Li, Shiqiang; Zheng, Mingqiang; Cai, Hancheng; Wang, Yongxian

2006-12-01

In an effort to develop a peptide-based radiopharmaceutical for the detection of tumors overexpressed vasoactive intestinal peptide receptors with positron emission tomography, we have prepared a novel [R(8,15,21), L17]-VIP peptide for 18F-labeling. This peptide inhibited 125I-VIP binding to rats lung membranes with high affinity [half-maximal inhibitory concentrations (IC50) of 0.12 nm]. Additionally, [R(8,15,21), L17]-VIP showed higher stability than native vasoactive intestinal peptide in vivo of mice. With N-succinimidyl 4-[18F] fluorobenzoate as labeling prosthetic group, [18F]FB-[R(8,15,21), L17]-VIP was obtained in >99% radiochemical purity within 100 min in decay-for-corrected radiochemical yield of 33.6 +/- 3% (n = 5) and a specific radioactivity 255 GBq/micromol at the end of synthesis. Stability of [18F]FB-[R(8,15,21), L17]-VIP in vitro and in vivo were investigated. Biodistribution of this trace was carried out in mice with induced C26 colorectal tumor. Fast clearance of [18F]FB-[R(8,15,21), L17]-VIP from non-target tissues and specific uptakes by tumors realized higher tumor-to-muscle ratio (3.55) and tumor-to-blood ratio (2.37) 60 min postinjection. Clear difference was observed between the blocking and unblocking experiments in biodistribution and whole body radioautography. [18F]FB-[R(8,15,21), L17]-VIP has demonstrated its potential for diagnosing tumors overexpressed vasoactive intestinal peptide receptors both in vitro and in vivo.

Large-area, uniform and low-cost dual-mode plasmonic naked-eye colorimetry and SERS sensor with handheld Raman spectrometer

NASA Astrophysics Data System (ADS)

Xu, Zhida; Jiang, Jing; Wang, Xinhao; Han, Kevin; Ameen, Abid; Khan, Ibrahim; Chang, Te-Wei; Liu, Gang Logan

2016-03-01

We demonstrated a highly-sensitive, wafer-scale, highly-uniform plasmonic nano-mushroom substrate based on plastic for naked-eye plasmonic colorimetry and surface-enhanced Raman spectroscopy (SERS). We gave it the name FlexBrite. The dual-mode functionality of FlexBrite allows for label-free qualitative analysis by SERS with an enhancement factor (EF) of 108 and label-free quantitative analysis by naked-eye colorimetry with a sensitivity of 611 nm RIU-1. The SERS EF of FlexBrite in the wet state was found to be 4.81 × 108, 7 times stronger than in the dry state, making FlexBrite suitable for aqueous environments such as microfluid systems. The label-free detection of biotin-streptavidin interaction by both SERS and colorimetry was demonstrated with FlexBrite. The detection of trace amounts of the narcotic drug methamphetamine in drinking water by SERS was implemented with a handheld Raman spectrometer and FlexBrite. This plastic-based dual-mode nano-mushroom substrate has the potential to be used as a sensing platform for easy and fast analysis in chemical and biological assays.We demonstrated a highly-sensitive, wafer-scale, highly-uniform plasmonic nano-mushroom substrate based on plastic for naked-eye plasmonic colorimetry and surface-enhanced Raman spectroscopy (SERS). We gave it the name FlexBrite. The dual-mode functionality of FlexBrite allows for label-free qualitative analysis by SERS with an enhancement factor (EF) of 108 and label-free quantitative analysis by naked-eye colorimetry with a sensitivity of 611 nm RIU-1. The SERS EF of FlexBrite in the wet state was found to be 4.81 × 108, 7 times stronger than in the dry state, making FlexBrite suitable for aqueous environments such as microfluid systems. The label-free detection of biotin-streptavidin interaction by both SERS and colorimetry was demonstrated with FlexBrite. The detection of trace amounts of the narcotic drug methamphetamine in drinking water by SERS was implemented with a handheld Raman spectrometer and FlexBrite. This plastic-based dual-mode nano-mushroom substrate has the potential to be used as a sensing platform for easy and fast analysis in chemical and biological assays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08357e

The Effects of Band Labels on Evaluators' Judgments of Musical Performance

ERIC Educational Resources Information Center

Silvey, Brian A.

2009-01-01

This study investigates the effects of band labels on evaluators' judgments of musical performance. High school concert band members (n = 72), wind ensemble members ( n = 77), and band directors (n = 8) were randomly assigned to a band label or no label group. Only the band label group was given evaluation forms that specified the group playing…

Vitamin D concentrations in fortified foods and dietary supplements intended for infants: Implications for vitamin D intake.

PubMed

Verkaik-Kloosterman, Janneke; Seves, S Marije; Ocké, Marga C

2017-04-15

Due to potential overages to cover losses during shelf life, the actual vitamin D concentration of fortified foods and dietary supplements may deviate from the label. In this pilot study the vitamin D concentrations of fortified foods (n=29; follow-on formula, baby porridge, curd cheese dessert) and dietary supplements (n=15), both specifically intended for infants, were analytically determined. Compared to the declared values, the vitamin D content ranged from 50% to 153% for fortified foods and from 8% to 177% for supplements. In general, both instant follow-on formula and oil-based supplements had a measured vitamin D content similar to or higher than the labelled value. Ready-to-eat baby porridge was the only category in which all measured vitamin D concentrations were below the declared value (74-81%). The use of label information for fortified foods and dietary supplements may result in invalid estimations of vitamin D intake distributions of infants; both under- and overestimation may occur. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Using the Triple Labelling Technique to apportion N2O Emissions to Nitrification and Denitrification from different Nitrogen Sources at different Water-Filled-Pore-Spaces

NASA Astrophysics Data System (ADS)

Loick, Nadine; Dixon, Elizabeth R.; Repullo Ruibérriz de Torres, Miguel A.; Ciganda, Veronica; Lopez-Aizpun, Maria A.; Matthews, G. Peter; Müller, Christoph; Cardenas, Laura M.

2017-04-01

Nitrous oxide (N2O) is considered to be an important greenhouse gas (GHG) accounting for approximately 6% of the current global warming. The atmospheric N2O concentration has been increasing since the Industrial Revolution, with soils representing its major source, making the understanding of its sources and removal processes very important for the development of mitigation strategies. In soils N gases are mainly produced via nitrification and denitrification. It is assumed that under dry/aerobic conditions nitrification is the dominant N consuming process, while denitrification becomes dominant under wetter conditions promoting anaerobicity. Nitrification and denitrification may occur simultaneously in different microsites of the same soil but there is often uncertainty associated with which process dominates in a particular soil under specific conditions. N2O predominantly derives from incomplete denitrification of nitrate (NO3-). The existence of different pools of NO3- in soils, namely the native soil pool, and the fertiliser-added one, has been suggested through a series of laboratory incubation experiments (Meijide et al., 2010; Bergstermann et al., 2011) using the denitrification incubation system, DENIS (Cardenas et al., 2003), in which soil cores are incubated under an N-free atmosphere, allowing direct measurements of all emitted N gases (NO, N2O and N2) as well as CO2. A third pool, NO3- produced from nitrification of applied NH4+, can also be a source of N2O via denitrification and also from nitrification. In this study labelling of substrate-N with 15N is used to quantify the underlying gross N transformation rates and link them to N-emissions to identify the production and consumption pathways and temporal dynamics of N2O. In three experiments twelve soil cores each were incubated in the DENIS to measure gaseous emissions, while parallel incubations under the same conditions were set up for destructive soil sampling at 7 time points. Using the triple labelling technique - i.e. applying NH4NO3 with either the N at the NH4+ or at the NO3-, or in both places being labelled - this study investigates the effects of a low, medium and high water filled pore space (55, 70, 85%) in a clay soil on gaseous N emissions and investigates the source and processes leading to N2O emissions. To assess the utilisation of applied NO3- vs nitrified NO3- from applied NH4+, the model developed by Müller et al. (2007) is used to calculate the immobilisation of added NO3- and NH4+, nitrification of added NH4+, mineralisation of organic N and subsequent nitrification by the analysis of the 15N in the soil. Gross transformation rates, indicating the relative importance of added NO3- and NO3- derived from nitrified added NH4+ are calculated. Bergstermann et al. (2011) Soil Biol. & Biochem. 43, 240-250. Meijide et al. (2010) Eur. J. Soil Sci. 61, 364-374. Cárdenas et al. (2003) Soil Biol. & Biochem. 35, 867-870. Müller et al. (2007) Soil Biol. & Biochem. 39, 715-726.

Ethnic label use in adolescents from traditional and non-traditional immigrant communities.

PubMed

Kiang, Lisa; Perreira, Krista M; Fuligni, Andrew J

2011-06-01

Understanding adolescents' use of ethnic labels is a key developmental issue, particularly given the practical significance of identity and self-definition in adolescents' lives. Ethnic labeling was examined among adolescents in the traditional immigrant receiving area of Los Angeles (Asian n = 258, Latino n = 279) and the non-traditional immigrant receiving area of North Carolina (Asian n = 165, Latino n = 239). Logistic regressions showed that adolescents from different geographic settings use different ethnic labels, with youth from NC preferring heritage and panethnic labels and youth from LA preferring hyphenated American labels. Second generation youth were more likely than first generation youth to use hyphenated American labels, and less likely to use heritage or panethnic labels. Greater ethnic centrality increased the odds of heritage label use, and greater English proficiency increased the odds of heritage-American label use. These associations significantly mediated the initial effects of setting. Further results examine ethnic differences as well as links between labels and self-esteem. The discussion highlights implications of ethnic labeling and context.

Nitrogen-rich higher-molecular soil organic compounds patterned by lignin degradation products: Considerations on the nature of soil organic nitrogen

NASA Astrophysics Data System (ADS)

Liebner, Falk; Bertoli, Luca; Pour, Georg; Klinger, Karl; Ragab, Tamer; Rosenau, Thomas

2016-04-01

The pathways leading to accumulation of covalently bonded nitrogen in higher-molecular soil organic matter (SOM) are still a controversial issue in soil science and geochemistry. Similarly, structural elucidation of the variety of the types of nitrogenous moieties present in SOM is still in its infancy even though recent NMR studies suggest amide-type nitrogen to form the majority of organically bonded nitrogen which is, however, frequently not in accordance with the results of wet-chemical analyses. Following the modified polyphenol theory of Flaig and Kononova but fully aware of the imperfection of a semi-abiotic simulation approach, this work communicates the results of a study that investigated some potential nitrogen accumulation pathways occurring in the re-condensation branch of the theory following the reactions between well-known low-molecular lignin and carbohydrate degradation products with nitrogenous nucleophiles occurring in soils under aerobic conditions. Different low-molecular degradation products of lignin, cellulose, and hemicellulose, such as hydroquinone, methoxyhydroquinone, p-benzoquinone, 2,5-dihydroxy-[1,4]benzoquinone, glucose, xylose, and the respective polysaccharides, i.e. cellulose, xylan as well as various types of lignin were subjected to a joint treatment with oxygen and low-molecular N-nucleophiles, such as ammonia, amines, and amino acids in aqueous conditions, partly using respective 15N labeled compounds for further 15N CPMAS NMR studies. Product mixtures derived from mono- and polysaccharides have been comprehensively fractionated and analyzed by GC/MS after derivatization. Some of ammoxidized polyphenols and quinones have been analyzed by X-ray photoelectron spectroscopy. Some products, such as those obtained from ammoxidation of methoxy hydroquinone using 15N labeled ammonia were fractionated following the IHSS protocol. Individual humin (H), humic acid (HA), and fulvic acid (FA) fractions were subjected to elemental analyses and NMR spectroscopy (1H, 13C, 15N), respectively. The reactions of hydroquinone, methyl hydroquinone and p-benzoquinone with ammonia and dimethyl-ammonium dimethylcarbamate - a source of dimethyl amine - affording the respective (substituted) 2,5-diamino-[1,4]benzoquinones under aerobic conditions have been studied by EPR spectroscopy for both oxygen-free and oxygen-rich conditions. 15N CPMAS NMR and XPS spectra of ammoxidized technical lignins and of ammoxidized low-molecular polyphenolic and quinoid products of the aerobic, microbial lignin degradation using 15N labeled aqueous ammonium hydroxide share many similarities, highly indicative for reaction sequences proceeding via common key intermediates. It has been demonstrated that 2,5-dihydroxy-[1,4]benzoquinone which can be surprisingly formed from both lignin and cellulose reacts with N nucleophiles to the respective 2,5-amino derivatives. The latter are semi-stable and react further to nitrogenous compounds of higher molecular weight. Hydroquinone and methoxy hydroquinone react even faster affording the respective 2,5-diamino-[1,4]benzoquinones. EPR experiments revealed that the reaction of hydroquinone with dimethyl amine proceed via radical intermediates. The results of this study strongly support the polyphenol theory and is hoped to contribute to a better understanding of nitrogen accumulation in soil organic matter. Acknowledgement The financial support by the Austrian Research Promotion Agency FFG (project FLIPPR: Future Lignin and Pulp Processing Research, 836650) and by The Austrian Science Fund FWF (project I154-N19) is gratefully acknowledged.

Using NanoSIMS coupled with microfluidics to visualize the early stages of coral infection by Vibrio coralliilyticus.

PubMed

Gibbin, E; Gavish, A; Domart-Coulon, I; Kramarsky-Winter, E; Shapiro, O; Meibom, A; Vardi, A

2018-04-20

Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral's capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature. Three coral fragments were inoculated with 15 N-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. 15 N-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative 15 N-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of 15 N-enriched hotspots, suggesting these tissues act as a "collection and digestion" site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these 15 N-hotspots revealed these to be host phagosomal and secretory cells/vesicles. This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

Hydrogen exchange kinetics in a membrane protein determined by sup 15 N NMR spectroscopy: Use of the INEPT (insensitive nucleus enhancement by polarization transfer) experiment to follow individual amides in detergent-solubilized M13 coat protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, G.D.; Sykes, B.D.

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous {sup 1}H nuclear magnetic resonance (NMR) study, multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow kinetic sets containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at leastmore » 10{sup 5}-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein the authors use {sup 15}N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiments can be used to transfer magnetization to the {sup 15}N nucleus from a coupled proton; when {sup 15}N-labeled protonated protein is dissolved in {sup 2}H{sub 2}O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H{sup +} and OH{sup {minus}} ions. The time-dependent exchange-out experiment is suitable for slow exchange rates (k{sub ex}). The INEPT experiment was also adapted to measure some of the more rapidly exchanging amides in the coat protein using either saturation transfer from water or exchange effects on the polarization transfer step itself. The results of all of these experiments are consistent with previous models of the coat protein in which a stable segment extends from the hydrophobic membrane-spanning region through to the C-terminus, whereas the N-terminal region is undergoing more extensive dynamic fluctuations.« less

Improving the design of nutrition labels to promote healthier food choices and reasonable portion sizes.

PubMed

Roberto, C A; Khandpur, N

2014-07-01

Accurate and easy-to-understand nutrition labeling is a worthy public health goal that should be considered an important strategy among many to address obesity and poor diet. Updating the Nutrition Facts Panel on packaged foods, developing a uniform front-of-package labeling system and providing consumers with nutrition information on restaurant menus offer important opportunities to educate people about food's nutritional content, increase awareness of reasonable portion sizes and motivate consumers to make healthier choices. The aims of this paper were to identify and discuss: (1) current concerns with nutrition label communication strategies; (2) opportunities to improve the communication of nutrition information via food labels, with a specific focus on serving size information; and (3) important future areas of research on nutrition labeling as a tool to improve diet. We suggest that research on nutrition labeling should focus on ways to improve food labels' ability to capture consumer attention, reduce label complexity and convey numeric nutrition information in simpler and more meaningful ways, such as through interpretive food labels, the addition of simple text, reduced use of percentages and easy-to-understand presentation of serving size information.

Hybrid Antibody-Induced Topographical Redistribution of Surface Immunoglobulins, Alloantigens, and Concanavalin A Receptors on Mouse Lymphoid Cells

PubMed Central

Stackpole, Christopher W.; De Milio, Lawrence T.; Hämmerling, Ulrich; Jacobson, Janet B.; Lardis, Michael P.

1974-01-01

Redistribution of surface immunoglobulins, H-2b, Thy-1.2, and TL.1,2,3 alloantigens, and concanavalin A receptors on mouse lymphoid cells induced by hybrid rabbit F(ab′)2 antibody (anti-mouse immunoglobulin/anti-visual marker or anti-concanavalin A/anti-visual marker) was studied by immunofluorescence. When used directly to label surface immunoglobulin, and indirectly to label alloantigens and concanavalin A receptors, hybrid antibodies induced similar displacement of all surface components from a uniform distribution into “patches” and “caps” at 37°. One hybrid antibody preparation, antimouse immunoglobulin/anti-ferritin, contained negligible amounts of bivalent anti-mouse immunoglobulin antibody, and was therefore “monovalent” for the antimouse immunoglobulin specificity. This observation suggests that factors other than multivalent crosslinking are responsible for hybrid antibody-induced redistribution of cell-surface components. Cap formation induced by hybrid antibody was enhanced markedly by attachment of the visual marker, either ferritin or southern bean mosaic virus, at 37°. At -5°, hybrid antibody does not displace uniformly distributed H-2b alloantigen-alloantibody complexes, but patches of label develop when ferritin attaches to the hybrid antibody. These results explain the patchy distribution of cell-surface components, which is a temperature-independent characteristic of labeling with hybrid antibodies and visual markers for electron microscopy. Images PMID:4595577

Localization of 15N uptake in a Tibetan alpine Kobresia pasture

NASA Astrophysics Data System (ADS)

Schleuß, Per-Marten; Kuzyakov, Yakov

2014-05-01

The Kobresia Pygmea ecotone covers approximately 450.000 km2 and is of large global and regional importance due several socio-ecological aspects. For instance Kobresia pastures store high amounts of carbon, nitrogen and other nutrients, represent large grazing areas for herbivores, provide a fast regrowth after grazing events and protect against mechanical degradation and soil erosion. However, Kobresia pastures are assumed to be a grazing induced and are accompanied with distinct root mats varying in thickness between 5-30 cm. Yet, less is known about the morphology and the functions of this root mats, especially in the background of a progressing degradation due to changes of climate and management. Thus we aimed to identify the importance of single soil layers for plant nutrition. Accordingly, nitrogen uptake from different soil depths and its remain in above-ground biomass (AGB), belowground biomass (BGB) and soil were determined by using a 15N pulse labeling approach during the vegetation period in summer 2012. 15N urea was injected into six different soil depths (0.5 cm, 2.5 cm, 7.5 cm, 12.5 cm, 17.5 cm, 22.5 cm / for each 4 replicates) and plots were sampled 45 days after the labeling. For soil and BGB samples were taken in strict sample intervals of 0-1 cm, 1-5 cm, 5-10 cm, 10-15 cm, 15-20 cm, 20-25 cm. Results indicate that total recovery (including AGB, BGB and soil) was highest, if tracer was injected into the top 5 cm and subsequently decreased with decreasing injection depth. This is especially the case for the 15N recovery of BGB, which is clearly attributed to the root density and strongly decreased with soil depth. In contrast, the root activity derived from the 15N content of roots increased with soil depth, which is primary associated to a proportionate increase of living roots related to dead roots. However, most 15N was captured in plant biomass (67.5-85.3 % of total recovery), indicating high 15N uptake efficiency possibly due to N limitation of Kobresia ecosystems. Considering only the nitrogen uptake of AGB hardly any differences appeared between the six injection depths. Nevertheless, it could be shown, that 50.4 % percent of total variance of AGB nitrogen uptake could be explained by combining root density and root activity. Concluding, from the upper root mat horizons highest amounts of nitrogen were taken up by plants, because root densities are correspondingly high. However, in deeper root mat layers the root activity increases and accordingly plays a key role for plant nitrogen supply in this depth. Underlying causes for increasing root activities may be better soil moisture conditions, lower variation of soil temperature and/or a higher access to plant available nitrogen in deeper soil layers.Please fill in your abstract text.

Measurement of Enzyme Isotope Effects.

PubMed

Kholodar, Svetlana A; Ghosh, Ananda K; Kohen, Amnon

2017-01-01

Enzyme isotope effects, or the kinetic effects of "heavy" enzymes, refer to the effect of isotopically labeled protein residues on the enzyme's activity or physical properties. These effects are increasingly employed in the examination of the possible contributions of protein dynamics to enzyme catalysis. One hypothesis assumed that isotopic substitution of all 12 C, 14 N, and nonexchangeable 1 H by 13 C, 15 N, and 2 H, would slow down protein picosecond to femtosecond dynamics without any effect on the system's electrostatics following the Born-Oppenheimer approximation. It was suggested that reduced reaction rates reported for several "heavy" enzymes accords with that hypothesis. However, numerous deviations from the predictions of that hypothesis were also reported. Current studies also attempt to test the role of individual residues by site-specific labeling or by labeling a pattern of residues on activity. It appears that in several systems the protein's fast dynamics are indeed reduced in "heavy" enzymes in a way that reduces the probability of barrier crossing of its chemical step. Other observations, however, indicated that slower protein dynamics are electrostatically altered in isotopically labeled enzymes. Interestingly, these effects appear to be system dependent, thus it might be premature to suggest a general role of "heavy" enzymes' effect on catalysis. © 2017 Elsevier Inc. All rights reserved.

Evaluation of a novel educational strategy, including inhaler-based reminder labels, to improve asthma inhaler technique.

PubMed

Basheti, Iman A; Armour, Carol L; Bosnic-Anticevich, Sinthia Z; Reddel, Helen K

2008-07-01

To evaluate the feasibility, acceptability and effectiveness of a brief intervention about inhaler technique, delivered by community pharmacists to asthma patients. Thirty-one pharmacists received brief workshop education (Active: n=16, CONTROL: n=15). Active Group pharmacists were trained to assess and teach dry powder inhaler technique, using patient-centered educational tools including novel Inhaler Technique Labels. Interventions were delivered to patients at four visits over 6 months. At baseline, patients (Active: 53, CONTROL: 44) demonstrated poor inhaler technique (mean+/-S.D. score out of 9, 5.7+/-1.6). At 6 months, improvement in inhaler technique score was significantly greater in Active cf. CONTROL patients (2.8+/-1.6 cf. 0.9+/-1.4, p<0.001), and asthma severity was significantly improved (p=0.015). Qualitative responses from patients and pharmacists indicated a high level of satisfaction with the intervention and educational tools, both for their effectiveness and for their impact on the patient-pharmacist relationship. A simple feasible intervention in community pharmacies, incorporating daily reminders via Inhaler Technique Labels on inhalers, can lead to improvement in inhaler technique and asthma outcomes. Brief training modules and simple educational tools, such as Inhaler Technique Labels, can provide a low-cost and sustainable way of changing patient behavior in asthma, using community pharmacists as educators.

Use of Novel Whole Core Incubations to Measure the Fate of Fertilizer N in a Flooded Agricultural System

NASA Astrophysics Data System (ADS)

Penton, C. R.; Bruland, G. L.; Popp, B. N.; Engstrom, P.; Tiedje, J.; Brown, G. A.; Deenik, J. L.

2010-12-01

We developed a new whole-core perfusion technique for tracking the fate of 15NH4+ added to intact vegetated cores. Taro plants (Colocasia esculenta) were field-grown in (20 cm diameter) cores for three months, which allowed exchange with natural porewater, then harvested. Following core extraction, surface and porewater were removed and 15NH4+ labeled porewater was slowly re-introduced to the core through a perfusion cap in the laboratory. Mini porewater equilibrators were placed in 1 cm increments through the sediment profile for porewater extraction during incubation. We also independently tested the ability of taro roots to oxygenate the subsurface by growing plants in nutrient agar and measuring O2 flux with a microelectrode. In the agar experiment, diurnal O2 transport was monitored and the application of wind across the taro leaves was found necessary to develop an oxygenated zone at the root tips. Using this information, the harvested taro were incubated in growth chambers after perfusion using three treatments: Vegetated without wind, vegetated with wind, and a non-vegetated control. Porewater was analyzed for 29+30N2, 15NH4+, 15NO3-, and unlabeled nitrate and ammonium species. Plant uptake of 15NH4+ was also determined. Quantitative PCR was performed on the sediment profiles of functional genes involved in nitrogen cycling for correlation to N transformations. The major pathway of N loss was root-mediated nitrification/denitrification followed by a flow of 29+30N2 through the aerenchyma. The vegetated wind treatment exhibited the highest concentrations of labeled N2 in the subsurface during all time periods. In contrast, the vegetated no wind treatment had much higher aerenchyma 29+30N2 concentration, accounting for ~100% of the subsurface N2 accumulation by day three of the incubation. Surface water N2 concentrations were also highest in the no wind treatment. After nine days the 29+30N2 concentrations dropped by ~70%, with little difference remaining among the treatments, indicating limitation by 15NH4+ diffusion. These results indicate that N2 is preferentially transported through the aerenchyma in taro and probably other plants grown in flooded agricultural fields. However, increased wind stress reduced transport through the aerenchyma and resulted in greater N2 accumulation in the subsurface, which indicates the importance of mass flow transport of air and its effect on oxygenation at the root tips. The results indicate that the complexity of N cycling in flooded agricultural systems may confound attempts to estimate in-situ N losses through porewater modeling, ‘classic’ isotope pairing techniques, or N flux chambers. The whole-core technique presented here allows for the measurement of multiple N pools and fates while minimizing system disturbance and more accurately representing field conditions.

The measurement of muscle protein synthesis in broilers with a flooding dose technique: use of 15N-labelled phenylalanine, GC-MS and GC-C-IRMS.

PubMed

Dänicke, S; Böttcher, W; Simon, O; Jeroch, H

2001-01-01

An experiment was carried out to measure fractional muscle protein synthesis rates (k(s)) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [15N]phenylalanine). K(s) was calculated from the [15N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period. The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [15N]APE and 0 to 0.62 [15N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [15N]APE and from 0.0004 to 0.0018 [15N]APE in GC-MS and GC-C-IRMS, respectively. K(s) was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K(s) in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation. It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [15N]phenylalanine, GC-MS and GC-C-IRMS.

Observations on preferential flow and horizontal transport of nitrogen fertilizer in the unsaturated zone

USGS Publications Warehouse

Wilkison, D.H.; Blevins, D.W.

1999-01-01

A study site underlain by a claypan soil was instrumented to examine the transport of fertilizer nitrogen (N) under corn (Zea mays L.) cultivation. The study was designed to examine N transport within the unsaturated zone and in interflow (the saturated flow of water on top of the claypan). A 15N- labeled fertilizer (labeled N), bromide (Br), and chloride (Cl) were used as field tracers. Rapid or prolonged infiltration events allowed water and dissolved solutes to perch on the claypan for brief periods. However, a well- developed network of preferential flow paths quickly diverted water and solutes through the claypan and into the underlying glacial till aquifer. Excess fertilizer N in the unsaturated zone supplied a continuous, but declining input of N to ground water for a period of 15 mo after a single fertilizer application. Calculated solute velocities through the claypan matrix (6.4 x 10-6 cm s-1) were similar to horizontal transport rates along the claypan (3.5 to 7.3 x 10-6 cm s-1) but much slower than infiltration rates determined for preferential flow paths (1.67 x 10-3 cm s-1). These flow paths accounted for 35% of the transport. A seasonally variable, dual mode of transport (matrix and preferential flow) prevented the claypan from being an effective barrier to vertical transport. Simulations of selected field observations, conducted using the variably saturated two- dimensional flow and transport model, VS2DT, confirmed the presence of a dual flow regime in the claypan.

Ingredient Consistency of Commercially Available Polyphenol and Tocopherol Nutraceuticals

PubMed Central

Remsberg, Connie M.; Good, Renee L.; Davies, Neal M.

2010-01-01

Label claims of vitamin E succinate and polyphenolic nutraceuticals are assessed. A validated HPLC method was utilized to assess vitamin E succinate products. Three novel LC/MS methods were used to assess the polyphenols, pterostilbene, phloretin, and myricetin, in dietary supplements. The amount of vitamin E succinate varied from 0-130% of the stated label content with two products containing vitamin E acetate rather than vitamin E succinate. Expected polyphenols were found in 7 of the 8 supplement products. None of the polyphenol supplements contained content within 100-120% of label claims. The present study indicates a lack of uniformity in nutraceutical products. PMID:27721342

Evaluation of a potential generator-produced PET tracer for cerebral perfusion imaging: Single-pass cerebral extraction measurements and imaging with radiolabeled Cu-PTSM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathias, C.J.; Welch, M.J.; Raichle, M.E.

1990-03-01

Copper(II) pyruvaldehyde bis(N4-methylthiosemicarbazone) (Cu-PTSM), copper(II) pyruvaldehyde bis(N4-dimethylthiosemicarbazone) (Cu-PTSM2), and copper(II) ethylglyoxal bis(N4-methylthiosemicarbazone) (Cu-ETSM), have been proposed as PET tracers for cerebral blood flow (CBF) when labeled with generator-produced 62Cu (t1/2 = 9.7 min). To evaluate the potential of Cu-PTSM for CBF PET studies, baboon single-pass cerebral extraction measurements and PET imaging were carried out with the use of 67Cu (t1/2 = 2.6 days) and 64Cu (t1/2 = 12.7 hr), respectively. All three chelates were extracted into the brain with high efficiency. There was some clearance of all chelates in the 10-50-sec time frame and Cu-PTSM2 continued to clear. Cu-PTSM andmore » Cu-ETSM have high residual brain activity. PET imaging of baboon brain was carried out with the use of (64Cu)-Cu-PTSM. For comparison with the 64Cu brain image, a CBF (15O-labeled water) image (40 sec) was first obtained. Qualitatively, the H2(15)O and (64Cu)-Cu-PTSM images were very similar; for example, a comparison of gray to white matter uptake resulted in ratios of 2.42 for H2(15)O and 2.67 for Cu-PTSM. No redistribution of 64Cu was observed in 2 hr of imaging, as was predicted from the single-pass study results. Quantitative determination of blood flow using Cu-PTSM showed good agreement with blood flow determined with H2(15)O. This data suggests that (62Cu)-Cu-PTSM may be a useful generator-produced radiopharmaceutical for blood flow studies with PET.« less

Maxwell AFB, Montgomery, Alabama. Revised Uniform Summary of Surface Weather Observations (RUSSWO)

DTIC Science & Technology

1974-09-19

VISION GAS . MAY 00-02 09 3,8 90 3,8 499 7,7 .1 11.6 3150 ___03.05, 1.1 4,0 1__ __ 4t, 15t6 139, *1 24#7 3188 0o.o13l ,5 397 __ 3t7 10,8 17,6 1__ 1 2497...WINDDIRECTION AND SPEED i (FROM HOURLY OBSERVATIONS) i ( ..1AXWELL AF B A-AA/HMM15C)MIRY 37m,72 sr-P STA TIONl STATIO lolUNK Tgllil "Ol N CLADA NOPAll (IS T

Redefining Molecular Amphipathicity in Reversing the "Coffee-Ring Effect": Implications for Single Base Mutation Detection.

PubMed

Huang, Chi; Wang, Jie; Lv, Xiaobo; Liu, Liu; Liang, Ling; Hu, Wei; Luo, Changliang; Wang, Fubing; Yuan, Quan

2018-05-21

The "coffee ring effect" is a natural phenomenon where sessile drops leave ring-shaped structures on solid surfaces upon drying. It drives non-uniform deposition of suspended compounds on substrates, which adversely affects many processes, including surface-assisted biosensing and molecular self-assembly. In this study, we describe how the coffee ring effect can be eliminated by controlling the amphipathicity of the suspended compounds, for example DNA modified with hydrophobic dye. Specifically, nuclease digestion of the hydrophilic DNA end converts the dye-labeled molecule into an amphipathic molecule (one with comparably weighted hydrophobic and hydrophilic ends) and reverses the coffee ring effect and results in uniform disc-shaped feature deposition of the dye. The amphipathic product decreases the surface tension of the sessile drops and induces Marangoni flow, which drives the uniform distribution of the amphipathic dye-labeled product in the drops. As proof-of-concept, this strategy was used in a novel enzymatic amplification method for biosensing to eliminate the coffee ring effect on a nitrocellulose membrane and increase assay reliability and sensitivity. Importantly, the reported strategy for eliminating the coffee ring effect can be extended to other sessile drop systems for potentially improving assay reliability, and sensitivity.

N-iodoacetyltyramine: Preparation and use in sup 125 I labeling by alkylation of sulfhydryl groups

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, C.M.; Mihal, K.A.; Krueger, R.J.

1989-06-01

Preparation and use of N-iodoacetyltyramine in generation of {sup 125}I-labeled compounds is described. The kinetics of alkylation of N-acetylcysteine by N-iodoacetyltyramine (k2 = 3.0 M-1 s-1) and N-chloroacetyltyramine (k2 = 0.12 M-1 s-1) indicate that N-iodoacetyltyramine is more useful for labeling sulfhydryl-containing compounds to high specific activity with {sup 125}I. Conditions for preparation of carrier-free {sup 125}I-labeled N-iodoacetyl-3-monoiodotyramine in 50% yield based on starting iodide are described. The high degree of group specificity of N-iodoacetyl-3-monoiodotyramine reaction with sulfhydryl groups is demonstrated by the high reactivity toward sulfhydryl-containing bovine serum albumin and low reactivity toward N-ethylmaleimide-blocked bovine serum albumin and IgG.more » {sup 125}I-labeled N-iodoacetyl-3-monoiodotyramine was also used to prepare an {sup 125}I-labeled ACTH derivative that retains full biological activity, further demonstrating the selectivity toward reactions with sulfhydryl groups.« less

Nitrogen accumulation and partitioning in a High Arctic tundra ecosystem from extreme atmospheric N deposition events.

PubMed

Choudhary, Sonal; Blaud, Aimeric; Osborn, A Mark; Press, Malcolm C; Phoenix, Gareth K

2016-06-01

Arctic ecosystems are threatened by pollution from recently detected extreme atmospheric nitrogen (N) deposition events in which up to 90% of the annual N deposition can occur in just a few days. We undertook the first assessment of the fate of N from extreme deposition in High Arctic tundra and are presenting the results from the whole ecosystem (15)N labelling experiment. In 2010, we simulated N depositions at rates of 0, 0.04, 0.4 and 1.2 g Nm(-2)yr(-1), applied as (15)NH4(15)NO3 in Svalbard (79(°)N), during the summer. Separate applications of (15)NO3(-) and (15)NH4(+) were also made to determine the importance of N form in their retention. More than 95% of the total (15)N applied was recovered after one growing season (~90% after two), demonstrating a considerable capacity of Arctic tundra to retain N from these deposition events. Important sinks for the deposited N, regardless of its application rate or form, were non-vascular plants>vascular plants>organic soil>litter>mineral soil, suggesting that non-vascular plants could be the primary component of this ecosystem to undergo measurable changes due to N enrichment from extreme deposition events. Substantial retention of N by soil microbial biomass (70% and 39% of (15)N in organic and mineral horizon, respectively) during the initial partitioning demonstrated their capacity to act as effective buffers for N leaching. Between the two N forms, vascular plants (Salix polaris) in particular showed difference in their N recovery, incorporating four times greater (15)NO3(-) than (15)NH4(+), suggesting deposition rich in nitrate will impact them more. Overall, these findings show that despite the deposition rates being extreme in statistical terms, biologically they do not exceed the capacity of tundra to sequester pollutant N during the growing season. Therefore, current and future extreme events may represent a major source of eutrophication. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Processing watershed-derived nitrogen in a well-flushed New England estuary

USGS Publications Warehouse

Tobias, C.R.; Cieri, M.; Peterson, B.J.; Deegan, Linda A.; Vallino, J.; Hughes, J.

2003-01-01

Isotopically labeled nitrate (15NO3-) was added continuously to the Rowley estuary, Massachusetts, for 22 d to assess the transport, uptake, and cycling of terrestrially derived nitrogen during a period of high river discharge and low phytoplankton activity. Isotopic enrichment of the 3.5-km tidal prism (150,000 m3) was achieved for the 3 weeks and allowed us to construct a nitrogen mass balance model for the upper estuary. Mean ??15NO3- in the estuary ranged from 300??? to 600???, and approximately 75%-80% of the 15N was exported conservatively as 15NO 3- to the coastal ocean. Essentially all of the 20%-25% of the 15N processed in the estuary occurred in the benthos and was evenly split between direct denitrification and autotrophic assimilation. The lack of water-column 15N uptake was attributed to low phytoplankton stocks and short water residence times (1.2-1.4 d). Uptake of water-column NO3- by benthic autotrophs (enriched in excess of 100???) was a function of NO3- concentration and satisfied up to 15% and 25% of the total nitrogen demand for benthic microalgae and macroalgae, respectively. Approximately 10% of tracer assimilated by benthic autotrophs was mineralized and released back to the water column as 15NH4+. By the end of the study, 15N storage in sediments and marsh macrophytes accounted for 50%-70% of the 15N assimilated in the estuary. These compartments may sequester watershed-derived nitrogen in the estuary for time scales of months to years.

Quantifying denitrification losses from a sub-tropical pasture in Queensland/Australia - use of the 15N gas flux method

NASA Astrophysics Data System (ADS)

Friedl, Johannes; Scheer, Clemens; Warner, Daniel; Grace, Peter

2014-05-01

The microbial mediated production of nitrous oxide (N2O) and its reduction to dinitrogen (N2) via denitrification represents a loss of nitrogen (N) from fertilised agro ecosystems to the atmosphere. Although denitrification remains a major uncertainty in estimating N losses from soils, the magnitude of N2 losses and related N2:N2O ratios from soils are largely unknown due to difficulties measuring N2 against a high atmospheric background. In order to address this lack of data, this study investigated the influence of different soil moisture contents on N2 and N2O emissions from a sub-tropical pasture in Queensland/Australia using the 15N gas flux method. Intact soil cores were incubated over 14 days at 80% and 100% water filled pore space (WFPS). Gas samples were taken up to six times per day after application of 15N labelled nitrate, equivalent to 50 kg N ha-1 and analysed for N2 and N2O by isotope ratio mass spectrometry. Fluxes were calculated assuming non-random 15N distribution in the headspace according to Mulvaney and Kurtz (1984) using the labelled pool of nitrate estimated from N2O measurements (Stevens and Laughlin 2001). The main product of denitrification in both treatments was N2. N2 emissions exceeded N2O emissions by a factor of 1.3 ± 0.3 at 80% WFPS and a factor of 3 ± 0.8 at 100% WFPS. The total amount of N-N2 lost over the incubation period was 13.5±1.0 kg N ha-1 at 80% WFPS and 21.8±1.8 kg ha-1 at 100% WFPS respectively. Over the entire incubation period, N2 emissions remained elevated at 100% WFPS, showing high variation between soil cores, while related N2O emissions decreased. At 80% WFPS, N2 emissions increased constantly over time showing significantly higher values after day five. At the same time, N2O fluxes declined. Consequently, N2:N2O ratios rose over the incubation period in both treatments. Overall denitrification rates and related N2:N2O ratios were higher at 100% WFPS compared to 80% WFPS, confirming WFPS as a major driver of denitrification. This study highlights denitrification as a major pathway of N loss for sub-tropical pasture systems with a substantial amount of applied fertiliser lost as N2 at high WFPS. The 15N gas flux method proved an effective tool in assessing N losses from fertilised soils. However, its suitability to determine N2 fluxes from soils with lower denitrification rates needs to be confirmed in future studies. The high variation between soil cores emphasises the need for field measurements with a high spatial and temporal resolution in order to capture the dynamics of N2 emissions. Mulvaney, R. L. and L. T. Kurtz. 1984. "Evolution of Dinitrogen and Nitrous Oxide from Nitrogen-15 Fertilized Soil Cores Subjected to Wetting and Drying Cycles1." Soil Sci. Soc. Am. J. 48 (3): 596-602. https://www.soils.org/publications/sssaj/abstracts/48/3/596. doi: 10.2136/sssaj1984.03615995004800030026x. Stevens, R. J. and R. J. Laughlin. 2001. "Lowering the detection limit for dinitrogen using the enrichment of nitrous oxide." Soil Biology and Biochemistry 33 (9): 1287-1289. http://www.sciencedirect.com/science/article/pii/S0038071701000360. doi: http://dx.doi.org/10.1016/S0038-0717(01)00036-0.

Large-area, uniform and low-cost dual-mode plasmonic naked-eye colorimetry and SERS sensor with handheld Raman spectrometer.

PubMed

Xu, Zhida; Jiang, Jing; Wang, Xinhao; Han, Kevin; Ameen, Abid; Khan, Ibrahim; Chang, Te-Wei; Liu, Gang Logan

2016-03-21

We demonstrated a highly-sensitive, wafer-scale, highly-uniform plasmonic nano-mushroom substrate based on plastic for naked-eye plasmonic colorimetry and surface-enhanced Raman spectroscopy (SERS). We gave it the name FlexBrite. The dual-mode functionality of FlexBrite allows for label-free qualitative analysis by SERS with an enhancement factor (EF) of 10(8) and label-free quantitative analysis by naked-eye colorimetry with a sensitivity of 611 nm RIU(-1). The SERS EF of FlexBrite in the wet state was found to be 4.81 × 10(8), 7 times stronger than in the dry state, making FlexBrite suitable for aqueous environments such as microfluid systems. The label-free detection of biotin-streptavidin interaction by both SERS and colorimetry was demonstrated with FlexBrite. The detection of trace amounts of the narcotic drug methamphetamine in drinking water by SERS was implemented with a handheld Raman spectrometer and FlexBrite. This plastic-based dual-mode nano-mushroom substrate has the potential to be used as a sensing platform for easy and fast analysis in chemical and biological assays.

Uniform deposition of size-selected clusters using Lissajous scanning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beniya, Atsushi; Watanabe, Yoshihide, E-mail: e0827@mosk.tytlabs.co.jp; Hirata, Hirohito

2016-05-15

Size-selected clusters can be deposited on the surface using size-selected cluster ion beams. However, because of the cross-sectional intensity distribution of the ion beam, it is difficult to define the coverage of the deposited clusters. The aggregation probability of the cluster depends on coverage, whereas cluster size on the surface depends on the position, despite the size-selected clusters are deposited. It is crucial, therefore, to deposit clusters uniformly on the surface. In this study, size-selected clusters were deposited uniformly on surfaces by scanning the cluster ions in the form of Lissajous pattern. Two sets of deflector electrodes set in orthogonalmore » directions were placed in front of the sample surface. Triangular waves were applied to the electrodes with an irrational frequency ratio to ensure that the ion trajectory filled the sample surface. The advantages of this method are simplicity and low cost of setup compared with raster scanning method. The authors further investigated CO adsorption on size-selected Pt{sub n} (n = 7, 15, 20) clusters uniformly deposited on the Al{sub 2}O{sub 3}/NiAl(110) surface and demonstrated the importance of uniform deposition.« less

Cardiac PET perfusion tracers: current status and future directions.

PubMed

Maddahi, Jamshid; Packard, René R S

2014-09-01

PET myocardial perfusion imaging (MPI) is increasingly being used for noninvasive detection and evaluation of coronary artery disease. However, the widespread use of PET MPI has been limited by the shortcomings of the current PET perfusion tracers. The availability of these tracers is limited by the need for an onsite ((15)O water and (13)N ammonia) or nearby ((13)N ammonia) cyclotron or commitment to costly generators ((82)Rb). Owing to the short half-lives, such as 76 seconds for (82)Rb, 2.06 minutes for (15)O water, and 9.96 minutes for (13)N ammonia, their use in conjunction with treadmill exercise stress testing is either not possible ((82)Rb and (15)O water) or not practical ((13)N ammonia). Furthermore, the long positron range of (82)Rb makes image resolution suboptimal and its low myocardial extraction limits its defect resolution. In recent years, development of an (18)F-labeled PET perfusion tracer has gathered considerable interest. The longer half-life of (18)F (109 minutes) would make the tracer available as a unit dose from regional cyclotrons and allow use in conjunction with treadmill exercise testing. Furthermore, the short positron range of (18)F would result in better image resolution. Flurpiridaz F 18 is by far the most thoroughly studied in animal models and is the only (18)F-based PET MPI radiotracer currently undergoing clinical evaluation. Preclinical and clinical experience with Flurpiridaz F 18 demonstrated a high myocardial extraction fraction, high image and defect resolution, high myocardial uptake, slow myocardial clearance, and high myocardial-to-background contrast that was stable over time-important properties of an ideal PET MPI radiotracer. Preclinical data from other (18)F-labeled myocardial perfusion tracers are encouraging. Copyright © 2014. Published by Elsevier Inc.

Triacylglycerol is produced from starch and polar lipids in the green alga Dunaliella tertiolecta.

PubMed

Pick, Uri; Avidan, Omri

2017-10-13

The halotolerant green alga Dunaliella tertiolecta accumulates starch and triacylglycerol (TAG) amounting to 70% and 10-15% of total cellular carbon, respectively, when exposed to nitrogen (N) deprivation. The purpose of this study was to clarify the inter-relationships between the biosynthesis of TAG, starch, and polar lipids (PLs) in this alga. Pulse labeling with [14C]bicarbonate was utilized to label starch and [14C]palmitic acid (PlA) to label lipids. Transfer of 14C into TAG was measured and used to calculate rates of synthesis. About two-thirds of the carbon in TAG originates from starch, and one-third is made de novo by direct CO2 assimilation. The level made from degradation of pre-formed PLs is estimated to be very small. Most of the de novo synthesis involves fatty acid transfer through PLs made during the first day of N deprivation. The results suggest that starch made by photosynthetic carbon assimilation at the early stages of N deprivation is utilized for synthesis of TAG. Trans-acylation from PLs is the second major contributor to TAG biosynthesis. The utilization of starch for TAG biosynthesis may have biotechnological applications to optimize TAG biosynthesis in algae. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Electroluminescence from InGaN/GaN multi-quantum-wells nanorods light-emitting diodes positioned by non-uniform electric fields.

PubMed

Park, Hyunik; Kim, Byung-Jae; Kim, Jihyun

2012-11-05

We report that the nanorod light-emitting diodes (LEDs) with InGaN/GaN multi-quantum-wells (MQWs) emitted bright electroluminescence (EL) after they were positioned and aligned by non-uniform electric fields. Firstly, thin film LED structures with MQWs on sapphire substrate were coated with SiO(2) nanospheres, followed by inductively-coupled plasma etch to create nanorod-shapes with MQWs, which were transferred to the pre-patterned SiO(2)/Si wafer. This method allowed us to obtain nanorod LEDs with uniform length, diameter and qualities. Dielectrophoretic force created by non-uniform electric field was very effective at positioning the processed nanorods on the pre-patterned contacts. After aligned by non-uniform electric field, we observed bright EL from many nanorods, which had both cases (p-GaN/MQWs/n-GaN or n-GaN/MQWs/p-GaN). Therefore, bright ELs at different locations were observed under the various bias conditions.

Tracing changes in soil N transformations to explain the doubling of N2O emissions under elevated CO2 in the Giessen FACE

NASA Astrophysics Data System (ADS)

Moser, Gerald; Brenzinger, Kristof; Gorenflo, Andre; Clough, Tim; Braker, Gesche; Müller, Christoph

2017-04-01

To reduce the emissions of greenhouse gases (CO2, CH4 & N2O) it is important to quantify main sources and identify the respective ecosystem processes. While the main sources of N2O emissions in agro-ecosystems under current conditions are well known, the influence of a projected higher level of CO2 on the main ecosystem processes responsible for N2O emissions has not been investigated in detail. A major result of the Giessen FACE in a managed temperate grassland was that a +20% CO2 level caused a positive feedback due to increased emissions of N2O to 221% related to control condition. To be able to trace the sources of additional N2O emissions a 15N tracing study was conducted. We measured the N2O emission and its 15N signature, together with the 15N signature of soil and plant samples. The results were analyzed using a 15N tracing model which quantified the main changes in N transformation rates under elevated CO2. Directly after 15N fertilizer application a much higher dynamic of N transformations was observed than in the long run. Absolute mineralisation and DNRA rates were lower under elevated CO2 in the short term but higher in the long term. During the one year study period beginning with the 15N labelling a 1.8-fold increase of N2O emissions occurred under elevated CO2. The source of increased N2O was associated with NO3- in the first weeks after 15N application. Elevated CO2 affected denitrification rates, which resulted in increased N2O emissions due to a change of gene transcription rates (nosZ/(nirK+nirS)) and resulting enzyme activity (see: Brenzinger et al.). Here we show that the reported enhanced N2O emissions for the first 8 FACE years do prevail even in the long-term (> 15 years). The effect of elevated CO2 on N2O production/emission can be explained by altered activity ratios within a stable microbial community.

Protein-based stable isotope probing.

PubMed

Jehmlich, Nico; Schmidt, Frank; Taubert, Martin; Seifert, Jana; Bastida, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Vogt, Carsten

2010-12-01

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.

Dissimilatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and effect of acetylene.

PubMed

Kaspar, H F; Tiedje, J M

1981-03-01

15N tracer methods and gas chromatography coupled to an electron capture detector were used to investigate dissimilatory reduction of nitrate and nitrite by the rumen microbiota of a fistulated cow. Ammonium was the only 15N-labeled end product of quantitative significance. Only traces of nitrous oxide were detected as a product of nitrate reduction; but in experiments with nitrite, up to 0.3% of the added nitrogen accumulated as nitrous oxide, but it was not further reduced. Furthermore, when 13NO3- was incubated with rumen microbiota virtually no [13N]N2 was produced. Acetylene partially inhibited the reduction of nitrite to ammonium as well as the formation of nitrous oxide. It is suggested that in the rumen ecosystem nitrous oxide is a byproduct of dissimilatory nitrite reduction to ammonium rather than a product of denitrification and that the latter process is absent from the rumen habitat.

Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.

PubMed Central

Hazum, E; Nimrod, A

1982-01-01

Photoaffinity labeling of rat ovarian granulosa cells and membrane preparations with a bioactive photoaffinity derivative of gonadotropin-releasing hormone resulted in identification of two specific components with apparent molecular weights of 60,000 and 54,000. Fluorescent visualization of gonadotropin-releasing hormone receptors in these cells, by using a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed patches that subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on the granulosa cells. These studies may provide an experimental basis for understanding the molecular events involved in the action of the hormone in the ovary. Images PMID:6281784

Impact of biochar application on nitrogen nutrition of rice, greenhouse-gas emissions and soil organic carbon dynamics in two paddy soils of China

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Zubin; Xu, Yanping; Liu, Gang

Two field microcosm experiments and 15N labeling techniques were used to investigate the first-year effects of biochar addition on rice N nutrition and GHG emissions in an Inceptisol and an Ultisol. Biochar N bioavailability and effect of biochar on fertilizer nitrogen-use efficiency (NUE) were studied by 15N-enriched wheat biochar (7.8803 atom% 15N) and fertilizer urea (5 atom% 15N) (Experiment I). Corn biochar and corn stalks were applied at 12 Mg ha-1 to study their effects on GHG emissions (Experiment II). Biochar had no significant impact on rice production and less than 2% of the biochar N was available to plantsmore » in the first season. Biochar addition increased soil C and N contents and decreased urea NUE.. Seasonal cumulative CH4 emissions with biochar were similar to the controls, but significantly lower than the local practice of straw amendment. Soil emissions of N2O with biochar amendment were similar to the control in the acidic Ultisol, but significantly higher in the slightly alkaline Inceptisol. Carbon-balance calculations found no major losses of biochar-C. Low bio-availability of biochar N did not make a significant impact on rice production or N nutrition during the first year.. Replacement of straw amendments with biochar could decrease CH4 emissions and increase SOC stocks.« less

Assessment of Apixaban Prescribing Patterns for Nonvalvular Atrial Fibrillation in Hospitalized Patients.

PubMed

Gibson, Caitlin M; Smith, Carmen B; Davis, Sondra; Scalese, Michael J

2018-01-01

Apixaban is a direct oral anticoagulant (DOAC) for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation (NVAF). Other DOACs require renal dose adjustments based solely on creatinine clearance. Apixaban differs in that its dose adjustments are more complex, potentially leading to prescribing errors. To determine if adherence to Food and Drug Administration (FDA)-approved dosing for apixaban is maintained in hospitalized patients with NVAF. Patients ≥18 years old with NVAF who received apixaban during admission to 1 of 3 hospitals were evaluated. The primary outcome was to determine if providers order apixaban in accordance with FDA-approved dosages. Secondary outcomes included determining if pharmacist review increased the number of orders in accordance with FDA-approved dosing, which of the 3 criteria (age ≥80 years, body weight ≤60 kg, or serum creatinine ≥1.5 mg/dL) were met in patients receiving off-label dosing, and the rationale for off-label prescribing. A total of 556 patients met inclusion criteria. Apixaban was dosed according to FDA labeling by providers in 83.4% (n = 464) of orders. After pharmacist review, 87.0% (n = 484) of orders were at the approved dose, 12.2% (n = 68) were underdosed, and 0.7% (n = 4) were overdosed. Most patients who were underdosed met only 1 dose reduction criterion-most commonly age ≥80 years (56.0%). Reasons for off-label dosing included home dose continuation (39.0%), history of or perceived bleeding risk (30.5%), or unspecified/other (30.5%). The majority of apixaban orders for NVAF were based on FDA-approved dosages after provider entry and pharmacist review.

[Screening with angiographic images prior to (99m)Tc-HMPAO labelled leukocyte scintigraphy in the diagnosis of periprosthetic infection].

PubMed

Granados, U; Fuster, D; Soriano, A; García, S; Bori, G; Martínez, J C; Mayoral, M; Perlaza, P; Tomás, X; Pons, F

2015-01-01

To evaluate the impact of the angioscintigrapy of the three phase bone scan as screening method to rule out infection of the hip and knee prosthesis prior to performing the (99m)Tc-HMPAO leukocyte scintigraphy. A total of 120 (70 women, 50 men; mean age 71±11years) with clinical suspicion of hip (n=63) or knee (n=57) infection of the prosthesis and clinical suspicion of infection were evaluated prospectively. All patients underwent three-phase bone scan (angioscintigraphy, vascular and bone phase) and (99m)Tc-HMPAO-labelled white blood cell scintigraphy. Final diagnosis of infection was made by microbiological documentation or clinical follow-up for at least 12months. Eighteen out of 120 patients were diagnosed of infection of hip prosthesis (n=10) or knee prosthesis (n=8). The angioscintigraphy was positive in 15/18 infected cases and in 21/102 of the non-infected cases with a sensitivity of 83%, specificity of 79% and negative predictive value of 97%. Sensitivity and specificity of (99m)Tc-HMPAO leukocyte scintigraphy were 72% and 95%, respectively. If the leukocyte labeled scintigraphies had been used exclusively for patients with positive angioscintigraphy, this would have saved up to 70% of the (99m)Tc-HMPAO leukocyte scintigraphies performed. There were no cases of infection with positive labeled leukocyte scintigraphy and negative angioscintigraphy. Angioscintigraphy (blood flow phase of bone scan) is a useful technique for screening for hip and knee joint prosthesis infection, significantly reducing the need for (99m)Tc-HMPAO leukocyte scintigraphy without affecting the sensitivity of the technique. Copyright © 2014 Elsevier España, S.L.U. and SEMNIM. All rights reserved.

Passage of stable isotope-labeled grass silage fiber and fiber-bound protein through the gastrointestinal tract of dairy cows.

PubMed

Warner, D; Dijkstra, J; Hendriks, W H; Pellikaan, W F

2013-01-01

Fractional passage rates are required to predict nutrient absorption in ruminants but data on nutrient-specific passage kinetics are largely lacking. With the use of the stable isotope ratio (δ) as an internal marker, we assessed passage kinetics of fiber and fiber-bound nitrogen (N) of intrinsically labeled grass silage from fecal and omasal excretion patterns of δ(13)C and δ(15)N. In a 6×6 Latin square, lactating dairy cows received grass silages [455 g/kg of total diet dry matter (DM) ] in a 2×3 factorial arrangement from ryegrass swards fertilized at low (45 kg of N/ha) or high (90 kg of N/ha) levels of N and harvested at 3 maturity stages. Feed intake (16.7±0.48 kg of DM/d; mean ± standard error of the mean) and milk yield (26.7±0.92 kg/d) increased at the high level of N fertilization and at decreasing maturity. Nutrient digestibility decreased with increasing plant maturity, particularly at the high level of N fertilization, essentially reflecting dietary treatment effects on the nutritional composition of the grass silage. Fractional rumen passage rates (K1) were highest and total mean retention time in the gastrointestinal tract (TMRT) was lowest when based on the external marker chromium mordanted fiber (Cr-NDF; 0.047/h and 38.0 h, respectively). Fecal δ(13)C in the acid detergent fiber fraction ((13)CADF) provided the lowest K1 (0.023/h) and the highest TMRT (61.1 h) and highest peak concentration time (PCT; 24.3h) among markers. In comparison, fecal fiber-bound N ((15)NADF) had a considerably higher K1 (0.032/h) and lower TMRT (46.4 h) than (13)CADF. Total N (measured with (15)NDM) had a comparable K1 (0.034/h) to that of (15)NADF but provided the highest fractional passage rates from the proximal colon-cecum (K2; 0.37/h) and lowest PCT (17.4 h) among markers. A literature review indicated unclear effects of grass silage maturity on K1 and unknown effects of N fertilization on K1. Our study indicated no effect of advancing maturity on fecal K1 and a trend for K1 to increase with the high level of N fertilization. Parameter K2 increased, whereas PCT and TMRT generally decreased with the high level of N fertilization. Omasal digesta sampling largely confirmed results based on fecal sampling. Results indicate that the use of δ(13)C and δ(15)N can describe fiber-specific passage kinetics of forage. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

78 FR 77656 - Aerosols and Similar Pressurized Containers-Meeting To Discuss the Method of Sale for Packages...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-24

..., Packaging and Labeling Regulation (UPLR) in NIST Handbook 130, ``Uniform Laws and Regulations in the Areas... permit price and quantity comparison is forbidden.'' Products using BOV technology versus traditional...

Creating 13C- and 15N-enriched tree leaf litter for decomposition experiments

NASA Astrophysics Data System (ADS)

Szlavecz, K. A.; Pitz, S.; Chang, C.; Bernard, M.

2013-12-01

Labeling plant material with heavy isotopes of carbon and nitrogen can produce a traceable nutrient signal that can be followed into the different trophic levels and decomposer food web. We treated 60 tree saplings with 13C-enriched CO2 gas and 15N-enriched ammonium nitrate over a three-month period to create dually-labeled plant material for future decomposition experiments. The trees included both early (Red maple, Sweetgum, Tulip poplar) and late (American beech, White oak) successional deciduous tree species, and a conifer, White pine. We constructed a 2.4 m × 2.4 m × 2.4 m environmental chamber that was climate-controlled using an air conditioning system. An Arduino microcontroller interfaced with a Vaisala GMP343 CO2 probe maintained a CO2 concentration between 500-520 ppm by controlling a solenoid valve on the CO2 tank regulator. The trees were placed into the chamber in August 2012 and remained until senescence unless they were lost to death or disease. Ammonium nitrate was added twice, in September and October. Leaf samples were collected prior to the start of the experiment and after senescence, whereas root samples were collected only in December. Samples were dried, ground and analyzed using an isotope ratio mass spectrometer. American beech and White oak had 40% mortality, and 34% of tulip poplar trees were removed because of powdery mildew overgrowth or death. Most tulip poplar trees exhibited a second leaf out following senescence in late September. Nearly 1 kg of litter was produced with tulip poplar representing over half of the total mass. Levels of enrichment varied greatly by species. Beech (-14.2‰) and White oak (-4.8‰) had low levels of enrichment in comparison to early successional species such as Sweetgum (41.7‰) and Tulip poplar (30.7‰ [first leaf fall] and 238.0‰ [second leaf fall]). Leaf enrichment with 15N followed a similar pattern, though it was achieved at a higher level with δ15N values varying from 271.6‰ to 1354.2‰ at the end of the experiment. Enrichment of roots was significantly higher than leaves (δ13C range: 111.5-219.2‰; δ15N range: 1516.9-3939.3‰) indicating that nutrients were translocated away from leaves prior to senescence, which is supported by the increase in C:N ratio between the initial (19.0) and final (60.1) leaf sampling. Despite the variable levels of enrichment, leaves from all species were sufficiently labeled for use in future studies aimed at tracking the transformation of carbon and nitrogen during decomposition. The greatest challenges were treating diseases and pests and creating ideal growing conditions for many species within the same chamber. Reducing the number of individuals and better pest management will lead to even higher level enrichment in the future.

Immunogold localisation of laminin in normal and exfoliative iris.

PubMed Central

Konstas, A. G.; Marshall, G. E.; Lee, W. R.

1990-01-01

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium. Images PMID:2390517

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

USGS Publications Warehouse

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

2017-01-01

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

Minot AFB, North Dakota. Revised Uniform Summary of Surface Weather Observations (RUSSWO). Parts A-F.

DTIC Science & Technology

1983-04-01

DEC I I. T! 8.61 57.9 6 1.5 21.1 1.3 19.8 37.9 71 TO~S8.3 2c.7t 2.1 26.8 .~5G* 18.A 1.? 8.% . 25. 8 43 USAFETAC 0 10 SIOL Al, P,.f usl 0-O .S n...N ih T m.p w.ato r. R[ .I. T0 F 32 F 6 F 73 IF 8 R P *93 F 1 ,To - Dry Bulb 4 W., SoIl 51 . -- - - -- - -- - - - - - ~ --- ----D..- - -~ GLOBAL

Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides

PubMed Central

Thakur, Chandar S.; Brown, Margaret E.; Sama, Jacob N.; Jackson, Melantha E.

2010-01-01

Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2813-y) contains supplementary material, which is available to authorized users. PMID:20730533

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics.

PubMed

Wang, Yang; Ruan, Qingyu; Lei, Zhi-Chao; Lin, Shui-Chao; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong

2018-04-17

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

Effects of Csk Homologous Kinase Overexpression on HER2/Neu-Mediated Signal Transduction Pathways in Breast Cancer Cells

DTIC Science & Technology

2005-04-01

several SH2 domains. The 5112 domains of CHE, Csk, Src, Lck, Shc, and phospholipase Cyl (PLCy1) were aligned using the T- COFFEE program (available at...iSC-Double-labeled protein samples or 15N_ acrylamide gel and then transferred onto Immobilon-PM membranes. bacteria in Bound proteins were

Uptake of 15N labeled fertilizer in loblolly pine plantations of the southern United States

Treesearch

Jay E. Raymond; Thomas Fox; Brian Strahm

2015-01-01

Forests in the southeastern United States managed extensively (minimal silvicultural treatments) generally have low productivity rates (Fox and others 2007). Conversely, intensively managed stands have significantly increased productivity in this region, from 2 m3 ha-1 year -1 to a range of 6 to 10 m3 ha-1 year -1 (Fox and others 2007).

A Comparison of Exogenous Labels for the Histological Identification of Transplanted Neural Stem Cells

PubMed Central

Nicholls, Francesca J.; Liu, Jessie R.; Modo, Michel

2017-01-01

The interpretation of cell transplantation experiments is often dependent on the presence of an exogenous label for the identification of implanted cells. The exogenous labels Hoechst 33342, 5-bromo-2′-deoxyuridine (BrdU), PKH26, and Qtracker were compared for their labeling efficiency, cellular effects, and reliability to identify a human neural stem cell (hNSC) line implanted intracerebrally into the rat brain. Hoechst 33342 (2 mg/ml) exhibited a delayed cytotoxicity that killed all cells within 7 days. This label was hence not progressed to in vivo studies. PKH26 (5 μM), Qtracker (15 nM), and BrdU (0.2 μM) labeled 100% of the cell population at day 1, although BrdU labeling declined by day 7. BrdU and Qtracker exerted effects on proliferation and differentiation. PKH26 reduced viability and proliferation at day 1, but this normalized by day 7. In an in vitro coculture assay, all labels transferred to unlabeled cells. After transplantation, the reliability of exogenous labels was assessed against the gold standard of a human-specific nuclear antigen (HNA) antibody. BrdU, PKH26, and Qtracker resulted in a very small proportion (<2%) of false positives, but a significant amount of false negatives (~30%), with little change between 1 and 7 days. Exogenous labels can therefore be reliable to identify transplanted cells without exerting major cellular effects, but validation is required. The interpretation of cell transplantation experiments should be presented in the context of the label's limitations. PMID:27938486

Label Review Training: Module 1: Label Basics, Page 15

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the consequences of improper labeling.

Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stols, L.; Sanville Millard, C.; Dementieva, I.

2004-03-01

A simplified approach developed recently for the production of heterologous proteins in Escherichia coli uses 2-liter polyethylene terephthalate beverage bottles as disposable culture vessels [Sanville Millard, C. et al. 2003. Protein Expr. Purif. 29, 311-320]. The method greatly reduces the time and effort needed to produce native proteins for structural or functional studies. We now demonstrate that the approach is also well suited for production of proteins in defined media with incorporation of selenomethionine to facilitate structure determination by multiwavelength anomalous diffraction. Induction of a random set of Bacillus stearothermophilus target genes under the new protocols generated soluble selenomethionyl proteinsmore » in good yield. Several selenomethionyl proteins were purified in good yields and three were subjected to amino acid analysis. Incorporation of selenomethionine was determined to be greater than 95% in one protein and greater than 98% in the other two. In the preceding paper [Zhao et al., this issue, pp. 87-93], the approach is further extended to production of [U-15N]- or [U-13C, U-15N]-labeled proteins. The approach thus appears suitable for high-throughput production of proteins for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy.« less

Biosynthesis and intracellular transport of the receptor for platelet-derived growth factor.

PubMed Central

Claesson-Welsh, L; Rönnstrand, L; Heldin, C H

1987-01-01

The biosynthesis of the receptor for platelet-derived growth factor (PDGF) was examined in metabolically labeled human foreskin fibroblasts. The receptor was synthesized as a 145-kDa precursor, which, when incubated with endo-beta-N-acetylglucosaminidase H (endo H), underwent a 15-kDa decrease in molecular mass. This indicates that the size of the core protein is about 130 kDa and that the 145-kDa form represents a receptor precursor carrying high-mannose N-linked oligosaccharide groups. Within 15 min after synthesis, the receptor was converted to a 165-kDa form. This form was entirely resistant to endo H treatment and probably represents a receptor molecule that has undergone further posttranslational modification, including O-linked glycosylation. Subsequently, within 30 min, a molecule of 170 kDa--i.e., the size of the mature receptor--appeared. A slightly larger molecule, of 175 kDa, which could be immunoprecipitated from PDGF-stimulated 32P-labeled cells, probably represents a receptor further modified by autophosphorylation. The 170-kDa molecule had an isoelectric point of about 4.5. Addition of PDGF increased the turnover rate of the 170-kDa PDGF receptor. Images PMID:2827155

Functionalized vertical GaN micro pillar arrays with high signal-to-background ratio for detection and analysis of proteins secreted from breast tumor cells.

PubMed

Choi, Mun-Ki; Kim, Gil-Sung; Jeong, Jin-Tak; Lim, Jung-Taek; Lee, Won-Yong; Umar, Ahmad; Lee, Sang-Kwon

2017-11-02

The detection of cancer biomarkers has recently attracted significant attention as a means of determining the correct course of treatment with targeted therapeutics. However, because the concentration of these biomarkers in blood is usually relatively low, highly sensitive biosensors for fluorescence imaging and precise detection are needed. In this study, we have successfully developed vertical GaN micropillar (MP) based biosensors for fluorescence sensing and quantitative measurement of CA15-3 antigens. The highly ordered vertical GaN MP arrays result in the successful immobilization of CA15-3 antigens on each feature of the arrays, thereby allowing the detection of an individual fluorescence signal from the top surface of the arrays owing to the high regularity of fluorophore-tagged MP spots and relatively low background signal. Therefore, our fluorescence-labeled and CA15-3 functionalized vertical GaN-MP-based biosensor is suitable for the selective quantitative analysis of secreted CA15-3 antigens from MCF-7 cell lines, and helps in the early diagnosis and prognosis of serious diseases as well as the monitoring of the therapeutic response of breast cancer patients.

Residue selective 15N CEST and CPMG experiments for studies of millisecond timescale protein dynamics.

PubMed

Niu, Xiaogang; Ding, Jienv; Zhang, Wenbo; Li, Qianwen; Hu, Yunfei; Jin, Changwen

2018-06-01

Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes on the millisecond timescale. Traditional pseudo 3D 15 N CEST and CPMG experiments have certain limitations in their applications. For example, both experiments have low sensitivity for broadened resonances, and the process of optimizing sample conditions and experimental parameters are often time consuming. To overcome these limitations, we herein present a new set of residue selective 15 N CEST and CPMG pulse sequences by employing the Hartmann-Hahn cross-polarization transfer of magnetization in both 1D and 2D schemes. Combined with frequency labeling in the indirect dimension using only a small number of increments, the pulse sequences in the 2D scheme can be applied on resonances in overlapped regions of the 1 H- 15 N HSQC spectrum. The pulse sequences were further applied on several proteins, demonstrating their advantages over the traditional CEST and CPMG experiments under specific circumstances. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of groundwater microorganisms capable of assimilating RDX-derived nitrogen during in-situ bioremediation.

PubMed

Cho, Kun-Ching; Fuller, Mark E; Hatzinger, Paul B; Chu, Kung-Hui

2016-11-01

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a nitroamine explosive, is commonly detected in groundwater at military testing and training sites. The objective of this study was to characterize the microbial community capable of using nitrogen derived from the RDX or RDX intermediates during in situ bioremediation. Active groundwater microorganisms capable of utilizing nitro-, ring- or fully-labeled (15)N-RDX as a nitrogen source were identified using stable isotope probing (SIP) in groundwater microcosms prepared from two wells in an aquifer previously amended with cheese whey to promote RDX biodegradation. A total of fifteen 16S rRNA gene sequences, clustered in Clostridia, β-Proteobacteria, and Spirochaetes, were derived from the (15)N-labeled DNA fractions, suggesting the presence of metabolically active bacteria capable of using RDX and/or RDX intermediates as a nitrogen source. None of the derived sequences matched RDX-degrading cultures commonly studied in the laboratory, but some of these genera have previously been linked to RDX degradation in site groundwater via (13)C-SIP. When additional cheese whey was added to the groundwater samples, 28 sequences grouped into Bacteroidia, Bacilli, and α-, β-, and γ-Proteobacteria were identified. The data suggest that numerous bacteria are capable of incorporating N from ring- and nitro-groups in RDX during anaerobic bioremediation, and that some genera may be involved in both C and N incorporation from RDX. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of nanoparticles as endocytic tracers in cellular microbiology

NASA Astrophysics Data System (ADS)

Zhang, Yuying; Hensel, Michael

2013-09-01

The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology.The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr01550e

Characteristics of drug use on sheep farms in Ontario, Canada

PubMed Central

Moon, Catherine S.; Berke, Olaf; Avery, Brent P.; McEwen, Scott A.; Reid-Smith, Richard J.; Scott, Lisa; Menzies, Paula

2010-01-01

This study examined characteristics of the use of drugs, especially antimicrobials, on Ontario sheep farms. Forty-nine sheep farms participated in a 12-month prospective study. Producers documented treatment events during the study period and drug use data from the records were summarized. The most frequently used drugs of the 15 drug categories used by producers belonged to the following categories: antimicrobial (40.7%, n = 2710), vitamin/mineral (12.0%), and biological (11.1%). Short-acting penicillin (27.2%, n = 1103), long-acting oxytetracycline (22.9%), and long-acting penicillin (21.9%) were the most frequently used antimicrobials. The drugs that were used most frequently on sheep farms were antimicrobials, of which 93% of treatments were extra-label. Extensive extra-label drug use may be the result of the limited number of drugs that are approved in Canada for use in sheep. PMID:21358930

Competitive immunoassay of phenobarbital by microchip electrophoresis with laser induced fluorescence detection.

PubMed

Huang, Yong; Zhao, Shulin; Shi, Ming; Liu, Jinwen; Liang, Hong

2011-05-23

A microchip electrophoresis method with laser induced fluorescence detection was developed for the immunoassay of phenobarbital. The detection was based on the competitive immunoreaction between analyte phenobarbital and fluorescein isothiocyanate (FITC) labeled phenobarbital with a limited amount of antibody. The assay was developed by varying the borate concentration, buffer pH, separation voltage, and incubation time. A running buffer system containing 35 mM borate and 15 mM sodium dodecyl sulfate (pH 9.5), and 2800 V separation voltage provided analysis conditions for a high-resolution, sensitive, and repeatable assay of phenobarbital. Free FITC-labeled phenobarbital and immunocomplex were separated within 30s. The calibration curve for phenobarbital had a detection limit of 3.4 nM and a range of 8.6-860.0 nM. The assay could be used to determine the phenobarbital plasma concentration in clinical plasma sample. Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of nutritional history on nitrogen assimilation in congeneric temperate and tropical scleractinian corals

USGS Publications Warehouse

Piniak, G.A.; Lipschultz, F.

2004-01-01

The nutritional history of corals is known to affect metabolic processes such as inorganic nutrient uptake and photosynthesis, but little is known about how it affects assimilation efficiency of ingested prey items or the partitioning of prey nitrogen between the host and symbiont. The temperate scleractinian coral Oculina arbuscula and its tropical congener Oculina diffusa were acclimated to three nutritional regimes (fed twice weekly, starved, starved with an inorganic nutrient supplement), then fed Artemia nauplii labeled with the stable isotope tracer 15N. Fed corals of both species had the lowest assimilation efficiencies (36-51% for O. arbuscula, 38-57% for O. diffusa), but were not statistically different from the other nutritional regimes. Fed and starved corals also had similar NU4+ excretion rates. This is inconsistent with decreased nitrogen excretion and reduced amino acid catabolism predicted by both the nitrogen recycling and conservation paradigms. In coral host tissue, ???90% of the ingested 15N was in the TCA-insoluble (protein and nucleic acids) and ethanol-soluble (amino acids/low molecular weight compounds) within 4 h of feeding. The TCA-insoluble pool was also the dominant repository of the label in zooxanthellae of both species (40-53% in O. arbuscula, 50-60% in O. diffusa). However, nutritional history had no effect on the distribution of prey 15N within the biochemical pools of the host or the zooxanthellae for either species. This result is consistent with the nitrogen conservation hypothesis, as preferential carbon metabolism would minimize the effects of starvation on nitrogen-containing biochemical pools. ?? Springer-Verlag 2004.

Ammonia fixation by humic substances: A nitrogen-15 and carbon-13 NMR study

USGS Publications Warehouse

Thorn, K.A.; Mikita, M.A.

1992-01-01

The process of ammonia fixation has been studied in three well characterized and structurally diverse fulvic and humic acid samples. The Suwannee River fulvic acid, and the IHSS peat and leonardite humic acids, were reacted with 15N-labelled ammonium hydroxide, and analyzed by liquid phase 15N NMR spectrometry. Elemental analyses and liquid phase 13C NMR spectra also were recorded on the samples before and after reaction with ammonium hydroxide. The largest increase in percent nitrogen occurred with the Suwannee River fulvic acid, which had a nitrogen content of 0.88% before fixation and 3.17% after fixation. The 15N NMR spectra revealed that ammonia reacted similarly with all three samples, indicating that the functional groups which react with ammonia exist in structural configurations common to all three samples. The majority of nitrogcn incorporated into the samples appears to be in the form of indole and pyrrole nitrogen, followed by pyridine, pyrazine, amide and aminohydroquinone nitrogen. Chemical changes in the individual samples upon fixation could not be discerned from the 13C NMR spectra.

Iron-59 absorption from soy hulls: intrinsic vs extrinsic labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lykken, G.I.; Mahalko, J.R.; Nielsen, E.J.

As part of an evaluation of the validity of the extrinsic labeling technique for measuring iron absorption, absorption from soy hulls extrinsically labeled (/sup 59/Fe added to bread dough) was compared with that from soy hulls intrinsically labeled (/sup 59/Fe incorporated into the soy plant during growth). Century soybeans were grown in a greenhouse. After pods had formed and were filling, each plant was stem injected twice, at 3 day intervals, with 22 ..mu..Ci /sup 59/Fe as FeCl/sub 2/ in 25 ..mu..l of 0.5 M HCl solution. After the plants had senesced, the soybeans were harvested, dried, shelled and themore » hulls removed. Standard meals containing 3.5 mg Fe/meal and up to 0.06 ..mu..Ci /sup 59/Fe in a soy hull bun were fed on 2 consecutive days to free-living volunteers in a crossover design. Absorption of /sup 59/Fe was greater from intrinsically labeled soy hulls than from extrinsically labeled soy hulls, 20 +/- 20% vs 15 +/- 11% (n=14, p > 0.05 by paired t-test). Apparent absorption ranged from 1.3% to 77% from intrinsically labeled soy hulls and .5% to 29% from extrinsically labeled soy hulls with the highest absorption occurring in persons with low serum ferritin (S.F. < 8 ng/ml). These findings provide additional evidence that the extrinsic labeling method is a valid measure of iron bioavailability to humans.« less

Catalytic Activity of Silicon Nanowires Decorated with Gold and Copper Nanoparticles Deposited by Pulsed Laser Ablation

PubMed Central

Casiello, Michele; Fusco, Caterina; Irrera, Alessia; Trusso, Sebastiano; Cotugno, Pietro

2018-01-01

Silicon nanowires (SiNWs) decorated by pulsed laser ablation with gold or copper nanoparticles (labeled as AuNPs@SiNWs and CuNPs@SiNWs) were investigated for their catalytic properties. Results demonstrated high catalytic performances in the Caryl–N couplings and subsequent carbonylations for gold and copper catalysts, respectively, that have no precedents in the literature. The excellent activity, attested by the very high turn over number (TON) values, was due both to the uniform coverage along the NW length and to the absence of the chemical shell surrounding the metal nanoparticles (MeNPs). A high recyclability was also observed and can be ascribed to the strong covalent interaction at the Me–Si interface by virtue of metal “silicides” formation. PMID:29385761

Catalytic Activity of Silicon Nanowires Decorated with Gold and Copper Nanoparticles Deposited by Pulsed Laser Ablation.

PubMed

Casiello, Michele; Picca, Rosaria Anna; Fusco, Caterina; D'Accolti, Lucia; Leonardi, Antonio Alessio; Lo Faro, Maria Josè; Irrera, Alessia; Trusso, Sebastiano; Cotugno, Pietro; Sportelli, Maria Chiara; Cioffi, Nicola; Nacci, Angelo

2018-01-30

Silicon nanowires (SiNWs) decorated by pulsed laser ablation with gold or copper nanoparticles (labeled as AuNPs@SiNWs and CuNPs@SiNWs) were investigated for their catalytic properties. Results demonstrated high catalytic performances in the C aryl -N couplings and subsequent carbonylations for gold and copper catalysts, respectively, that have no precedents in the literature. The excellent activity, attested by the very high turn over number (TON) values, was due both to the uniform coverage along the NW length and to the absence of the chemical shell surrounding the metal nanoparticles (MeNPs). A high recyclability was also observed and can be ascribed to the strong covalent interaction at the Me-Si interface by virtue of metal "silicides" formation.