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A method for efficient isotopic labeling of recombinant proteins  

Microsoft Academic Search

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H\\/13C\\/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled

Jonathan Marley; Min Lu; Clay Bracken



Isotope-labeled immunoassays without radiation waste  

E-print Network

Isotope-labeled immunoassays without radiation waste Guomin Shan*, Wei Huang*, Shirley J. Gee with radioactive materials, and (iii) short shelf-life of the labeled re- agents. The advantage of isotopic with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay

Hammock, Bruce D.


Glutathione specifically labeled with isotopes  

SciTech Connect

A procedure for synthesis of glutathione selectivity labeled with isotopes is described. A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques is immobilized in a carrageenan matrix and treated with toluene to render the cells more permeable to the substrates. The immobilized cell matrix is incubated with a mixture containing the appropriately labeled amino acid, the other amino acid constituents of glutathione, ATP, and acetylphosphate. The radiolabeled product is isolated by column chromatography.

Murata, K.; Abbott, W.A.; Bridges, R.J.; Meister, A.



Isotope Labeling in Insect Cells  

PubMed Central

Recent years have seen remarkable progress in applying nuclear magnetic resonance (NMR) spectroscopy to proteins that have traditionally been difficult to study due to issues with folding, posttranslational modification, and expression levels or combinations thereof. In particular, insect cells have proved useful in allowing large quantities of isotope-labeled, functional proteins to be obtained and purified to homogeneity, allowing study of their structures and dynamics by using NMR. Here, we provide protocols that have proven successful in such endeavors. PMID:22167667

Saxena, Krishna; Dutta, Arpana; Klein-Seetharaman, Judith



Simple, rapid method for the preparation of isotopically labeled formaldehyde  


Isotopically labeled formaldehyde (*C.sup..sctn.H.sub.2O) is prepared from labeled methyl iodide (*C.sup..sctn.H.sub.3I) by reaction with an oxygen nucleophile having a pendant leaving group. The mild and efficient reaction conditions result in good yields of *C.sup..sctn.H.sub.2O with little or no *C isotopic dilution. The simple, efficient production of .sup.11CH.sub.2O is described. The use of the .sup.11CH.sub.2O for the formation of positron emission tomography tracer compounds is described. The reaction can be incorporated into automated equipment available to radiochemistry laboratories. The isotopically labeled formaldehyde can be used in a variety of reactions to provide radiotracer compounds for imaging studies as well as for scintillation counting and autoradiography.

Hooker, Jacob Matthew (Port Jefferson, NY); Schonberger, Matthias (Mains, DE); Schieferstein, Hanno (Aabergen, DE); Fowler, Joanna S. (Bellport, NY)



Multiple isotopic labels for quantitative mass spectrometry  

PubMed Central

Quantitative mass spectrometry is often performed using isotopically-labeled samples. While the 4-trimethylammoniumbutyryl (TMAB) labels have many advantages over other isotopic tags, only two forms have previously been synthesized (i.e. a heavy form containing 9 deuteriums and a light form without deuterium). In the present report, two additional forms containing 3 and 6 deuteriums have been synthesized and tested. These additional isotopic tags perform identically to the previously reported tags; peptides labeled with the new TMAB reagents co-elute from reverse phase HPLC columns with peptides labeled with the lighter and heavier TMAB reagents. Altogether, these 4 tags allow for multivariate analysis in a single liquid chromatography/mass spectrometry analysis, with each isotopically tagged peptide differing in mass by 3 Da per tag incorporated. The synthetic scheme is described in simple terms so that a biochemist without specific training in organic chemistry can perform the synthesis. The interpretation of tandem mass spectrometry data for the TMAB-labeled peptides is also described in more detail. The additional TMAB isotopic reagents described here, together with the additional description of the synthesis and analysis should allow these labels to be more widely used for proteomics and peptidomics analyses. PMID:19551992

Morano, Cain; Zhang, Xin; Fricker, Lloyd D.



Catalytic gasification: Isotopic labeling and transient reaction  

SciTech Connect

Temperature-programmed reaction was used with labeled isotopes (/sup 13/C and /sup 18/O) to study interactions between carbon black and potassium carbonate in pure He and 10% CO/sub 2//90% He atmospheres. Catalytic gasification precursor complexes were observed. Carbon and oxygen-bearing carbon surface groups interacted with the carbonate above 500 K to form surface complexes. Between 500 K and 950 K, and in the presence of gaseous carbon dioxide, the complexes promoted carbon and oxygen exchange between the gas-phase CO/sub 2/ and the surface. Oxygen exchanged between the surface complexes; but carbon did not exchange between the carbonate and the carbon black. As the temperature rose, the complexes decomposed to produce carbon dioxide, and catalytic gasification then began. Elemental potassium formed, and the active catalyst appears to alternate between potassium metal and a potassium-oxygen-carbon complex.

Saber, J.M.; Falconer, J.L.; Brown, L.F.



Analysis of proteome dynamics in mice by isotopic labeling.  


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

Price, John C; Ghaemmaghami, Sina



ICPLQuant - A software for non-isobaric isotopic labeling proteomics.  


The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope-coded protein label (ICPL)-labeled peptides on the MS level during LC-MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time-consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS-identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker. PMID:19953540

Brunner, Achim; Keidel, Eva-Maria; Dosch, Dominik; Kellermann, Josef; Lottspeich, Friedrich



Quantitative Analysis of Snake Venoms Using Soluble Polymer-based Isotope Labeling*S?  

PubMed Central

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting ability, and fibrinogenolytic activities of crude venoms were measured and correlated with difference in protein abundance determined by the SoPIL analysis. The SoPIL approach could provide an efficient and widely applicable tool for quantitative proteomics. PMID:18089550

Galan, Jacob A.; Guo, Minjie; Sanchez, Elda E.; Cantu, Esteban; Rodriguez-Acosta, Alexis; Perez, John C.; Tao, W. Andy



Quantitative proteomics using reductive dimethylation for stable isotope labeling.  


Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample. PMID:25045933

Tolonen, Andrew C; Haas, Wilhelm



Raman spectroscopic and mass spectrometric investigations of the hydrogen isotopes and isotopically labelled methane  

Microsoft Academic Search

Suitable analytical methods must be tested and developed for monitoring the individual process steps within the fuel cycle of a fusion reactor and for tritium accountability. The utility of laser-Raman spectroscopy accompanied by mass spectrometry with an Omegatron was investigated using the analysis of all hydrogen isotopes and isotopically labeled methanes as an example. The Omegatron is useful for analyzing

Fluor Daniel Hanford



Kinetics of Precursor Labeling in Stable Isotope Labeling in Cell Cultures (SILAC) Experiments.  


Recent advances in mass spectrometry have enabled proteome-wide analyses of cellular protein turnover. These studies have been greatly propelled by the development of stable isotope labeling in cell cultures (SILAC), a set of standardized protocols, reagents aimed at quantifying the incorporation of (15)N/(13)C labeled amino acids into proteins. In dynamic SILAC experiments, the degree of isotope incorporation in proteins is measured over time and used to determine turnover kinetics. However, the kinetics of isotope incorporation in proteins can potentially be influenced not only by their intracellular turnover but also by amino acid uptake, recycling and aminoacyl-tRNA synthesis. To assess the influence of these processes in dynamic SILAC experiments, we have measured the kinetics of isotopic enrichment within intracellular free amino acid and aminoacyl-tRNA precursor pools in dividing and division-arrested neuroblastoma cells following the introduction of extracellular (15)N labeled amino acids. We show that the total flux of extracellular amino acids into cells greatly exceeds that of intracellular amino acid recycling and synthesis. Furthermore, in comparison to internal sources, external amino acids are preferentially utilized as substrates for aminoacyl-tRNA precursors for protein synthesis. As a result, in dynamic SILAC experiments conducted in culture, the aminoacyl-tRNA precursor pool is near completely labeled in a few hours and protein turnover is the limiting factor in establishing the labeling kinetics of most proteins. PMID:25301408

Zhang, Tian; Price, John C; Nouri-Nigjeh, Eslam; Li, Jun; Hellerstein, Marc K; Qu, Jun; Ghaemmaghami, Sina



Quantifying Peptides in Isotopically Labeled Protease Digests by Ion Mobility/Time-of-Flight  

E-print Network

Quantifying Peptides in Isotopically Labeled Protease Digests by Ion Mobility/Time-of-Flight Mass of isotopically labeled peptides. The isotopic labels were generated by treatment of peptides with N amines (i.e., the N-terminus and lysine residues). The relative concentrations of a peptide in each

Clemmer, David E.


Site-specific isotopic labeling of proteins for NMR studies  

SciTech Connect

NMR spectroscopy is a sensitive, site-specific probe of biomolecular structure. For relatively small proteins and peptides, the H resonances can be assigned using the sequential method. However, there are many cases, especially larger proteins, in which the spectra are too complex for complete, systematic resonance assignments. In some cases, assignments can be made by selective isotopic labeling (e.g., uniform incorporation of a {sup 13}C-labeled amino acid) in conjunction with site-directed mutagenesis or {sup 13}C, {sup 15}N double labeling of adjacent amino acids. However, in many large proteins, protein complexes, and unfolded proteins, resonance overlap and broadening prevents assignments. The ability to synthesize proteins with unnatural amono acids, beyond those specified by the genetic code, makes it possible to isotopically label a single amino acid residue in a protein. We report here the use of this approach to generate a T4 lysozyme (T4L) mutant containing a unique {sup 13}C-labeled alanine, for which {sup 13}C-filtered proton spectra were obtained in both the native and denatured states. This general methodology should be applicable to a variety of NMR measurements in large proteins and protein complexes. 14 refs., 1 fig.

Ellman, J.A.; Volkman, B.F.; Mendel, D. [Univ. of California, Berkeley, CA (United States)] [and others



18O Stable Isotope Labeling in MS-based Proteomics  

PubMed Central

A variety of stable isotope labeling techniques have been developed and used in mass spectrometry (MS)-based proteomics, primarily for relative quantitation of changes in protein abundances between two compared samples, but also for qualitative characterization of differentially labeled proteomes. Differential 16O/18O coding relies on the 18O exchange that takes place at the C-terminal carboxyl group of proteolytic fragments, where two 16O atoms are typically replaced by two 18O atoms by enzyme-catalyzed oxygen-exchange in the presence of H218O. The resulting mass shift between differentially labeled peptide ions permits identification, characterization and quantitation of proteins from which the peptides are proteolytically generated. This review focuses on the utility of 16O/18O labeling within the context of mass spectrometry-based proteome research. Different strategies employing 16O/18O are examined in the context of global comparative proteome profiling, targeted subcellular proteomics, analysis of post-translational modifications and biomarker discovery. Also discussed are analytical issues related to this technique, including variable 18O exchange along with advantages and disadvantages of 16O/18O labeling in comparison with other isotope-coding techniques. PMID:19151093

Ye, Xiaoying; Luke, Brian; Andresson, Thorkell



From isotope labeled CH3CN to N2 inside single-walled carbon nanotubes  

E-print Network

From isotope labeled CH3CN to N2 inside single-walled carbon nanotubes Christian Kramberger to this peculiar place? We have used N15 and C13 isotope labeled acetonitrile during the synthesis of single-walled carbon nanotubes to investigate this process. The isotope shifts of phonons and vibrons are observed

Maruyama, Shigeo


Quantitating isotopic molecular labels with accelerator mass spectrometry.  


Accelerator mass spectrometry (AMS) traces isotopically labeled biochemicals and provides significant new directions for understanding molecular kinetics and dynamics in biological systems. AMS traces low-abundance radioisotopes for high specificity but detects them with MS for high sensitivity. AMS reduces radiation exposure doses to levels safe for use in human volunteers of all ages. Total radiation exposures are equivalent to those obtained in very short airplane flights, a commonly accepted radiation risk. Waste products seldom reach the Nuclear Regulatory Commission (NRC) definition of radioactive waste material for (14)C and (3)H. Attomoles of labeled compounds are quantified in milligram-sized samples, such as 20 microl of blood. AMS is available from several facilities that offer services and new spectrometers that are affordable. Detailed examples of designing AMS studies are provided, and the methods of analyzing AMS data are outlined. PMID:16401517

Vogel, John S; Love, Adam H



Quantification of Isotopically Overlapping Deamidated and 18O Labeled Peptides Using Isotopic Envelope Mixture Modeling  

PubMed Central

A robust peptide quantification method was developed where overlapping peptide isotopic distributions were fit with predicted peptide isotopic envelope mixture models (IEMM). Application to two difficult quantitative problems was demonstrated. The first was the quantification of deamidation, where masses of isotopic peaks differ by a single Da, and the second, 18O labeling, where the isotopic peaks are shifted 2 and 4 Da. In both cases, peptide quantification cannot be performed by simple integration of extracted ion chromatograms, because the isotopic envelopes of mass shifted peptides are normally not resolved. To test the methodology for quantification of deamidation, several synthetic peptides and their corresponding deamidated forms were mixed at various ratios (1:0, 1:2, 2:1, 4:1, 10:1 and 20:1) and analyzed using the IEMM method, resulting in a high correlation (R2=0.96) between measured and known percentages of deamidation. The IEMM method was then incorporated into a workflow for deamidation quantification in a large-scale proteomics experiment. A series of normal (3-day, 2-year, 35-year, and 70-year) and cataractous (93-year) human lenses were analyzed using two-dimensional liquid chromatography tandem mass spectrometry and deamidation quantities of several ?S-crystallin peptides ([N14-Q16], N53, [Q63–Q70], and N143) were determined. Two peptides (N53 and [Q63–Q70]) had more extensive deamidation in the water-insoluble portions of normal lens samples, and deamidation at N143 was more extensive in the 93-year water-insoluble cataractous sample. The utility of the technique for analysis of 18O-labeled peptides was examined using mixtures of labeled BSA peptides in known 16O to 18O ratios (10:1, 4:1, 1:1, 1:4, and 1:10). The methodology allowed accurate measurements of ratios of 16O/18O peptides over the wide range of relative abundances. PMID:19173613

Dasari, Surendra; Wilmarth, Phillip A.; Reddy, Ashok P.; Robertson, Lucinda J. G.; Nagalla, Srinivasa R.; David, Larry L.



Relative quantitation of glycopeptides based on stable isotope labeling using MALDI-TOF MS.  


We have developed an effective, sensitive method for quantitative glycopeptide profiling using stable isotope labeling and MALDI-TOF mass spectrometry (MS). In this study, we synthesized benzoic acid-d0 N-succinimidyl ester (BzOSu) and benzoic acid-d5 N-succinimidyl ester (d-BzOSu) as light and heavy isotope reagents for stable isotope quantification for the comparative analysis of glycopeptides. Using this approach provided enhanced ionization efficiency in both positive and negative modes by MALDI-TOF MS. These reagents were quantitatively reacted with glycopeptides from human serum IgG (hIgG) at a wide range of concentrations; the labeling efficiency of the glycopeptides showed high reproducibility and a good calibration curve was obtained. To demonstrate the practical utility of this approach, we characterized the structures of glycopeptides from hIgG and from IgG1 produced by myeloma plasma. The glycopeptides were quantitatively analyzed by mixing Bz-labeled IgG1 glycopeptides with d-Bz-labeled hIgG glycopeptides. Glycan structural identification of the hIgG glycopeptides was demonstrated by combining the highly specific recognition of endo-?-N-acetyl glucosaminidases from Streptococcus pyogenes (endoS) or from Streptococcus pneumoniae (endo-D) with MALDI-TOF MS analysis. The obtained data revealed the glycan profile and the ratio of glycan structural isomers containing a galactosylated extension on IgG1, IgG2 and IgG3 glycopetides. PMID:25010467

Kurogochi, Masaki; Amano, Junko



Mathematical modeling of isotope labeling experiments for metabolic flux analysis.  


Isotope labeling experiments (ILEs) offer a powerful methodology to perform metabolic flux analysis. However, the task of interpreting data from these experiments to evaluate flux values requires significant mathematical modeling skills. Toward this, this chapter provides background information and examples to enable the reader to (1) model metabolic networks, (2) simulate ILEs, and (3) understand the optimization and statistical methods commonly used for flux evaluation. A compartmentalized model of plant glycolysis and pentose phosphate pathway illustrates the reconstruction of a typical metabolic network, whereas a simpler example network illustrates the underlying metabolite and isotopomer balancing techniques. We also discuss the salient features of commonly used flux estimation software 13CFLUX2, Metran, NMR2Flux+, FiatFlux, and OpenFLUX. Furthermore, we briefly discuss methods to improve flux estimates. A graphical checklist at the end of the chapter provides a reader a quick reference to the mathematical modeling concepts and resources. PMID:24218213

Nargund, Shilpa; Sriram, Ganesh



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

PubMed Central

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

Wasylenko, Thomas M.; Stephanopoulos, Gregory



Autotrophic Production of Stable-Isotope-Labeled Arginine in Ralstonia eutropha Strain H16  

PubMed Central

With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong PcbbL promoter of the R. eutropha H16 cbbc operon (coding for autotrophic CO2 fixation). In batch cultures growing on H2 and CO2 as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with 13CO2 and 15NH4Cl, and the 13C/15N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [13C/15N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%. PMID:22941075

Lutte, Steffen; Pohlmann, Anne; Zaychikov, Evgeny; Becher, Johannes R.; Heumann, Hermann; Friedrich, Barbel



A Novel Method for Relative Quantitation of N-Glycans by Isotopic Labeling Using 18O-Water  

PubMed Central

Quantitation is an essential aspect of comprehensive glycomics study. Here, a novel isotopic-labeling method is described for N-glycan quantitation using 18O-water. The incorporation of the 18O-labeling into the reducing end of N-glycans is simply and efficiently achieved during peptide-N4-(N-acetyl-?-glucosaminyl) asparagine amidase F release. This process provides a 2-Da mass difference compared with the N-glycans released in 16O-water. A mathematical calculation method was also developed to determine the 18O/16O ratios from isotopic peaks. Application of this method to several standard glycoprotein mixtures and human serum demonstrated that this method can facilitate the relative quantitation of N-glycans over a linear dynamic range of two orders, with high accuracy and reproducibility.

Tao, Shujuan; Orlando, Ron



A rapid method to attain isotope labeled small soluble peptides for NMR studies.  


A widely applicable strategy is presented for efficient and rapid production of small water soluble peptides expressed as fusion proteins with the immunoglobulin-binding domain of streptococcal protein G. A simple extraction and purification scheme that includes a protease cleavage step to release the target peptide is described. The yield of authentic target peptide exceeds 10 mg per liter of culture. Production of U-13C, 15N and highly deuterated U-13C, 15N isotope labeled peptide is demonstrated for the 11 residue S2 peptide, corresponding to the C-terminus of the alpha-subunit of transducin, and the coiled coil trimerization domain from cartilage matrix protein (CMPcc), respectively. Heteronuclear two-dimensional NMR spectra are used for initial peptide characterization. PMID:12766417

Koenig, Bernd W; Rogowski, Marco; Louis, John M



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

PubMed Central

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

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



Relative quantification of biomarkers using mixed-isotope labeling coupled with MS  

PubMed Central

The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers. PMID:23157360

Chapman, Heidi M; Schutt, Katherine L; Dieter, Emily M; Lamos, Shane M



Energy-efficient appliance labeling in China: Lessons for successful labeling programs in varied markets  

SciTech Connect

Appliance ownership and production has increased dramatically in China in the past two decades. From extremely low levels in 1980, China's appliance industry has become one of the largest in the world, with sales topping U.S. $14.4 billion in 2000. In 1981, less than 1 percent of urban Chinese households owned a refrigerator; by 1998, that number had increased to over 75 percent. This dramatic increase in sales and ownership leads to an excellent opportunity to impact energy consumption in China by affecting the energy efficiency of appliances being bought and sold. In general, Chinese consumers value energy efficiency and are knowledgeable about the operating costs of major appliances. However, the Chinese marketplace does not provide information that consumers trust about the energy consumption of specific products. Thus, several interdependent organizations have emerged in China to provide information and market supports for energy efficiency. This paper describes the appliance market in China and the evolution of its standards and labeling programs and the agencies that implement them. It discusses the authors' work with these organizations in developing energy efficiency criteria and supporting an energy efficiency endorsement labeling program in China. It describes how the authors have used their experience with ENERGY STAR{reg_sign} and other programs in the U.S. to work with China to develop a successful program specific to Chinese conditions, with a particular emphasis on refrigerators. It then gives the author's market assessment of the Chinese refrigerator market and recommendations for a successful labeling program and transferable lessons for developing energy efficiency labeling programs in varied markets. This paper is based on the authors' market research, their support in setting energy efficiency criteria in China, interviews with Chinese manufacturers, retailers, and sales staff, and the development and implementation of labeling strategies and promotion in China.

Lin, Jiang; Townend, Jeanne; Fridley, David; McNeil, Gary; Silva, Tony; Clark, Robin



Profiling of thiol-containing compounds by stable isotope labeling double precursor ion scan mass spectrometry.  


Here we developed a novel strategy of isotope labeling in combination with high-performance liquid chromatography-double precursor ion scan mass spectrometry (IL-LC-DPIS-MS) analysis for nontargeted profiling of thiol-containing compounds. In this strategy, we synthesized a pair of isotope labeling reagents (?-bromoacetonylquinolinium bromide, BQB; ?-bromoacetonylquinolinium-d7 bromide, BQB-d7) that contain a reactive group, an isotopically labeled moiety, and an ionizable group to selectively label thiol-containing compounds. The BQB and BQB-d7 labeled compounds can generate two characteristic product ions m/z 218 and 225, which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. The peak pairs with characteristic mass differences can be readily extracted from the two precursor ion scan (PIS) spectra and assigned as potential thiol-containing candidates, which facilitates the identification of analytes. BQB and BQB-d7 labeled thiol-containing compounds can be clearly distinguished by generating two individual ion chromatograms. Thus, thiol-containing compounds from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, offering good identification and accurate quantification by eliminating the MS response fluctuation and mutual interference from the two labeled samples. Using the IL-LC-DPIS-MS strategy, we profiled the thiol-containing compounds in beer and human urine, and 21 and 103 thiol candidates were discovered in beer and human urine, respectively. In addition, 9 and 17 thiol candidates in beer and human urine were successfully identified by further comparison with thiol standards or tandem mass spectrometry analysis. Taken together, the IL-LC-DPIS-MS method is demonstrated to be a promising strategy in the profiling of compounds with identical groups in metabolomics study. PMID:25222826

Liu, Ping; Huang, Yun-Qing; Cai, Wen-Jing; Yuan, Bi-Feng; Feng, Yu-Qi



Studies on peptide acetylation for stable-isotope labeling after 1-D PAGE separation in quantitative proteomics.  


Acetylation is a single labeling process to label peptides in control and experimental samples universally, and is independent of amino acid composition or post-translational modification. Here, we propose a new strategy especially useful to quantify either hydrophobic or extremely acidic and basic proteins involved in acetylation of tryptic peptides after sodium dodecyl sulfate polyarcylamide gel electrophoresis (SDS-PAGE) separation. We studied some essential parameters of acetylation labeling reactions in either in-solution tryptic peptides or in-gel digested extracts systematically. We have found that the acetylation efficiency varies markedly on account of different reactive systems, and demonstrated that stable isotope labeling can be steadily obtained with in-gel digested peptides under optimized conditions. We use this protocol to quantify some proteins of two kinds of hepatocellular carcinoma cell line, non-metastatic hepatocellular carcinoma cells, Hep3B, and metastatic hepatocellular carcinoma cells, MHCC97-H. The experimental results provide positive evidence for the potential application of an acetylation labeling strategy in quantitative proteomics, and an efficient way for global proteome quantification. PMID:15378704

Yu, Yanling; Cui, Jiefeng; Wang, Xiaoyan; Liu, Yinkun; Yang, Pengyuan



Radioactive labeling of antibody: a simple and efficient method  

Microsoft Academic Search

A simple and efficient method of covalently coupling the strong chelator diethylenetriaminepentaacetic acid to proteins was developed for radiolabeling immunoglobulin G antibodies. After being coupled and labeled with indium-111, a monoclonal antibody to carcinoembryonic antigen retained its ability to bind to its antigen in vitro and in vivo. In nude mice with a human colorectal xenograft, 41 percent of the

D. J. Hnatowich; W. W. Layne; R. L. Childs; D. Lanteigne; M. A. Davis; T. W. Griffin; P. W. Doherty



An experimental evaluation of transgenerational isotope labelling in a coral reef grouper  

Microsoft Academic Search

Transgenerational isotope labelling (TRAIL) using enriched stable isotopes provides a novel means of mass-marking marine fish\\u000a larvae and estimating larval dispersal. The technique, therefore, provides a new way of addressing questions about demographic\\u000a population connectivity and larval export from no-take marine protected areas. However, successful field applications must\\u000a be preceded by larval rearing studies that validate the geochemical marking technique,

David H. Williamson; Geoffrey P. Jones; Simon R. Thorrold



Structural determination of larger proteins using stable isotope labeling and NMR spectroscopy  

SciTech Connect

The project sought to employ stable isotope labeling and NMR spectroscopy to study protein structures and provide insight into important biochemical problems. A methylotrophic bacterial expression system has been developed for uniform deuterium and carbon-13 labeling of proteins for structural studies. These organisms grow using methanol as the sole source of carbon and energy. Because isotopically labeled methanol is relatively inexpensive, the methylotrophs are ideal for expressing proteins labeled uniformly with deuterium and/or carbon-13. This expression system has been employed to prepare deuterated troponin C. NMR spectroscopy measurements have been made on the inhibitory peptide from troponin I (residues 96--115), both as the free peptide and the peptide complexed with deuterated troponin C. Proton-NMR spectroscopy resonance-signal assignments have been made for the free peptide.

Unkefer, C.; Hernandez, G.; Springer, P.; Trewhella, J. [Los Alamos National Lab., NM (United States); Blumenthal, D. [Univ. of Utah, Salt Lake City, UT (United States); Lidstrom, M. [California Inst. of Tech., Pasadena, CA (United States)



A new method for the labelling of proteins with radioactive arsenic isotopes  

NASA Astrophysics Data System (ADS)

Radioarsenic labelled radiopharmaceuticals could be a valuable asset to positron emission tomography. In particular, the long half-lives of 72As ( T=26 h) and 74As ( T=17.8 d) allow to investigate slow physiological or metabolical processes, like the enrichment and distribution of monoclonal antibodies (mab) in tumour tissue. In this work, a new method for the labelling of proteins with various radioactive arsenic isotopes was developed. For this purpose, two proteins, namely a chimeric IgG 3 monoclonal antibody, ch3G4, directed against anionic phospholipids, and Rituxan (Rituximab), were labelled as a proof of principle with no-carrier-added radioarsenic isotopes ( 74As and 77As). The developed labelling chemistry gives high yields (>99.9%), is reliable and could easily be transferred to automated labelling systems in a clinical environment. At least for the mab used in this work, this route of radioarsenic labelling does not affect the immunoreactivity of the product. The arsenic label stays stable for up to 72 h at the molecular mass of the monoclonal antibody, which is in particular relevant to follow the pharmacology and pharmacokinetics of the labelled mab for several days.

Jennewein, M.; Hermanne, A.; Mason, R. P.; Thorpe, P. E.; Rösch, F.



A free-air system for long-term stable carbon isotope labeling of adult forest trees  

EPA Science Inventory

Stable carbon (C) isotopes, in particular employed in labeling experiments, are an ideal tool to broaden our understanding of C dynamics in trees and forest ecosystems. Here, we present a free-air exposure system, named isoFACE, designed for long-term stable C isotope labeling in...


Extensive backbone dynamics in the GCAA RNA tetraloop analyzed using 13C NMR spin relaxation and specific isotope labeling  

PubMed Central

Conformational dynamics play a key role in the properties and functions of proteins and nucleic acids. Heteronuclear NMR spin relaxation is a uniquely powerful site-specific probe of dynamics in proteins and has found increasing applications to nucleotide base side chains and anomeric sites in RNA. Applications to the nucleic acid ribose backbone, however, have been hampered by strong magnetic coupling among ring carbons in uniformly 13C-labeled samples. In this work, we apply a recently-developed, metabolically-directed isotope labeling scheme that places 13C with high efficiency and specificity at the nucleotide ribose C2’ and C4’ sites. We take advantage of this scheme to explore backbone dynamics in the well-studied GCAA RNA tetraloop. Using a combination of CPMG (Carr-Purcell-Meiboom-Gill) and R1? relaxation dispersion spectroscopy to explore exchange processes on the microsecond to millisecond timescale, we find an extensive pattern of dynamic transitions connecting a set of relatively well-defined conformations. In many cases, the observed transitions appear to be linked to C3’-endo/C2’-endo sugar pucker transitions of the corresponding nucleotides, and may also be correlated across multiple nucleotides within the tetraloop. These results demonstrate the power of NMR spin relaxation based on alternate-site isotope labeling to open a new window into the dynamic properties of ribose backbone groups in RNA. PMID:19049467

Johnson, James E.; Hoogstraten, Charles G.



Stable Isotope Labeling in Zebrafish Allows in Vivo Monitoring of Cardiac Morphogenesis*  

PubMed Central

Quantitative proteomics is an important tool to study biological processes, but so far it has been challenging to apply to zebrafish. Here, we describe a large scale quantitative analysis of the zebrafish proteome using a combination of stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS). Proteins derived from the fully labeled fish were used as a standard to quantify changes during embryonic heart development. LC-MS-assisted analysis of the proteome of activated leukocyte cell adhesion molecule zebrafish morphants revealed a down-regulation of components of the network required for cell adhesion and maintenance of cell shape as well as secondary changes due to arrest of cellular differentiation. Quantitative proteomics in zebrafish using the stable isotope-labeling technique provides an unprecedented resource to study developmental processes in zebrafish. PMID:23412571

Konzer, Anne; Ruhs, Aaron; Braun, Helene; Jungblut, Benno; Braun, Thomas; Kruger, Marcus



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

PubMed Central

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

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



Use of oxygen-18 isotopic labeling to assay photorespiration in terrestrial plants and algae  

SciTech Connect

A new method was devised to quantify photorespiration. The assay utilized {sup 18}O{sub 2} to isotopically label intermediates of the glycolate pathway, specifically glycolate, glycine, and serine, for various time periods. The pathway intermediates were isolated and analyzed on a mass spectrometer to determine molecular percent {sup 18}O-enrichment. Rates of glycolate synthesis were determined from: {sup 18}O-labeling kinetics of the intermediates, derived rate equations, and non-linear regression techniques. The method was adapted to measure photorespiratory rates in both terrestrial plants and algae. Test plants are Triticum aestivum, Zea mays L., Pavlova lutheri and Chlorella pyrenoidosa.

de Veau, E.J.



Synthesis of isotopically labeled 1,3-dithiane.  


The 1,3-dithiane is a protected formaldehyde anion equivalent that could serve as a useful labeled synthon. We report a facile synthesis of 1,3-[2-(13)C]- and 1,3-[2-(13)C, 2-(2)H2]dithiane in two steps from [(13)C]- or [(13) C, (2)H3 ]methyl phenyl sulfoxide. We have previously reported the high yield synthesis of [(13)C]methyl phenyl sulfide from [(13)C]MEOH and the oxidation of [(13)C]methyl phenyl sulfide to [(13)C]methyl phenyl sulfoxide. Here, we describe the facile exchange of deuterium from (2) H2 O into [(13)C]methyl phenyl sulfoxide to yield [(13)C, (2)H3]methyl phenyl sulfoxide. Thus, from [(13)C]MEOH and (2)H2O, all possible C2 stable isotopomers of 1,3-dithiane are available. Our synthetic route is also amenable to preparation of radiolabeled 1,3-dithianes. PMID:24861982

Martinez, Rodolfo A; Glass, David R; Ortiz, Erick G; Alvarez, Marc A; Unkefer, Clifford J



Radioactive Labeling of Antibody: A Simple and Efficient Method  

NASA Astrophysics Data System (ADS)

A simple and efficient method of covalently coupling the strong chelator diethylenetriaminepentaacetic acid to proteins was developed for radiolabeling immunoglobulin G antibodies. After being coupled and labeled with indium-111, a monoclonal antibody to carcinoembryonic antigen retained its ability to bind to its antigen in vitro and in vivo. In nude mice with a human colorectal xenograft, 41 percent of the injected radioactivity became localized in each gram of xenograft at 24 hours compared with 9 percent for control antibody and 19 percent for radioiodinated antibody to carcinoembryonic antigen.

Hnatowich, D. J.; Layne, W. W.; Childs, R. L.; Lanteigne, D.; Davis, M. A.; Griffin, T. W.; Doherty, P. W.



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


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

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



Identification of miRNA targets with stable isotope labeling by amino acids in cell culture  

Microsoft Academic Search

miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection. This repressed set of genes significantly overlaps with miRNA-1 regulated genes that have been

Jeppe Vinther; Mads M. Hedegaard; Paul P. Gardner; Jens S. Andersen; Peter Arctander



UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling  

SciTech Connect

We present UNiquant, a new software program for analyzing stable isotope labeling (SIL) based quantitative proteomics data. UNiquant surpassed the performance of two other platforms, MaxQuant and Mascot Distiller, using complex proteome mixtures having either known or unknown heavy/light ratios. UNiquant is compatible with a broad spectrum of search engines and SIL methods, providing outstanding peptide pair identification and accurate measurement of the relative peptide/protein abundance.

Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven; Fu, Kai; Ding, Shi-Jian



An Efficient Method to Estimate Labelled Sample Size for Transductive LDA(QDA/MDA)  

E-print Network

An Efficient Method to Estimate Labelled Sample Size for Transductive LDA(QDA/MDA) Based on Bayes to Estimate Labelled Sample Size 275 kernel, and by maximizing the margin based on the unlabelled data labelled sample size becomes a necessity. Moreover, a detailed analysis of labelled sample size under


Mass-related inversion symmetry breaking and phonon self-energy renormalization in isotopically labeled AB-stacked bilayer graphene  

E-print Network

A mass-related symmetry breaking in isotopically labeled bilayer graphene (2LG) was investigated during in-situ electrochemical charging of AB stacked (AB-2LG) and turbostratic (t-2LG) layers. The overlap of the two ...

Araujo, Paulo Antonio Trinidade


Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes  

Microsoft Academic Search

Abstact  A reaction of high-temperature solid-phase catalytic isotope exchange (HSCIE) was studied for the preparation of tritium-\\u000a and deuterium-labeled ligands of glutamate and dopamine receptors. Tritium-labeled (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclopenten-5,1-imine ([G-3H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-3H]-7-OH-DPAT) were obtained with a specific activity of 210 and 120 Ci\\/mol, respectively. The isotopomeric distribution of\\u000a deuterium-labeled ligands was studied using time-of-flight mass-spectrometer MX 5310 (ESI-o-TOF) with electrospray and orthogonal

Yu. A. Zolotarev; Yu. Yu. Firsova; A. Abaimov; A. K. Dadayan; V. S. Kosik; A. V. Novikov; N. V. Krasnov; B. V. Vaskovskii; I. V. Nazimov; G. I. Kovalev; N. F. Myasoedov



Molecular and mass spectroscopic analysis of isotopically labeled organic residues  

NASA Technical Reports Server (NTRS)

Experimental studies aimed at understanding the evolution of complex organic molecules on interstellar grains were performed. The photolysis of frozen gas mixtures of various compositions containing H2O, CO, NH3, and CH4 was studied. These species were chosen because of their astrophysical importance as deducted from observational as well as theoretical studies of ice mantles on interstellar grains. These ultraviolet photolyzed ices were warmed up in order to produce refractory organic molecules like the ones formed in molecular clouds when the icy mantles are being irradiated and warmed up either by a nearby stellar source or impulsive heating. The laboratory studies give estimates of the efficiency of production of such organic material under interstellar conditions. It is shown that the gradual carbonization of organic mantles in the diffuse cloud phase leads to higher and higher visual absorptivity - yellow residues become brown in the laboratory. The obtained results can be applied to explaining the organic components of comets and their relevance to the origin of life.

Mendoza-Gomez, Celia X.; Greenberg, J. Mayo; Mccain, P.; Ferris, J. P.; Briggs, R.; Degroot, M. S.; Schutte, Willem A.



Efficient methods and practical guidelines for simulating isotope effects.  


The shift in chemical equilibria due to isotope substitution is frequently exploited to obtain insight into a wide variety of chemical and physical processes. It is a purely quantum mechanical effect, which can be computed exactly using simulations based on the path integral formalism. Here we discuss how these techniques can be made dramatically more efficient, and how they ultimately outperform quasi-harmonic approximations to treat quantum liquids not only in terms of accuracy, but also in terms of computational cost. To achieve this goal we introduce path integral quantum mechanics estimators based on free energy perturbation, which enable the evaluation of isotope effects using only a single path integral molecular dynamics trajectory of the naturally abundant isotope. We use as an example the calculation of the free energy change associated with H/D and (16)O/(18)O substitutions in liquid water, and of the fractionation of those isotopes between the liquid and the vapor phase. In doing so, we demonstrate and discuss quantitatively the relative benefits of each approach, thereby providing a set of guidelines that should facilitate the choice of the most appropriate method in different, commonly encountered scenarios. The efficiency of the estimators we introduce and the analysis that we perform should in particular facilitate accurate ab initio calculation of isotope effects in condensed phase systems. PMID:23298033

Ceriotti, Michele; Markland, Thomas E



Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics  

Microsoft Academic Search

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultane- ous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of

Shao-En Ong; Blagoy Blagoev; Irina Kratchmarova; Dan Bach Kristensen; Hanno Steen; Akhilesh Pandey; Matthias Mann



Selectively Dispersed Isotope Labeling for Protein Structure Determination by Magic Angle Spinning NMR  

PubMed Central

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-13C] acetate does not label ? carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-13C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra. PMID:23990199

Eddy, Matthew T.; Belenky, Marina; Sivertsen, Astrid; Griffin, Robert G.; Herzfeld, Judith




SciTech Connect

The isotopic effect of palladium has been applied in different ways to separate hydrogen isotopes for many years. At Savannah River Site palladium deposited on kieselguhr (Pd/k) is used in a thermal cycling absorption process (TCAP) to purify tritium for over ten years. The need to design columns for different throughputs and the desire to advance the performance of TCAP created the need to evaluate different column designs and packing materials for their separation efficiency. In this work, columns with variations in length, diameter and metal foam use, were tested using an isotope displacement method. A simple computer model was also developed to calculate the number of theoretical separation stages using the test results. The effects of column diameter, metal foam and gas flow rate were identified.

Heung, L; Gregory Staack, G; James Klein, J; William Jacobs, W



Chemical imaging of biological materials by NanoSIMS using isotopic and elemental labels  

SciTech Connect

The NanoSIMS 50 combines unprecedented spatial resolution (as good as 50 nm) with ultra-high sensitivity (minimum detection limit of {approx}200 atoms). The NanoSIMS 50 incorporates an array of detectors, enabling simultaneous collection of 5 species originating from the same sputtered volume of a sample. The primary ion beam (Cs{sup +} or O{sup -}) can be scanned across the sample to produce quantitative secondary ion images. This capability for multiple isotope imaging with high spatial resolution provides a novel new approach to the study of biological materials. Studies can be made of sub-regions of tissues, mammalian cells, and bacteria. Major, minor and trace element distributions can be mapped on a submicron scale, growth and metabolism can be tracked using stable isotope labels, and biogenic origin can be determined based on composition. We have applied this technique extensively to mammalian and prokaryotic cells and bacterial spores. The NanoSIMS technology enables the researcher to interrogate the fate of molecules of interest within cells and organs through elemental and isotopic labeling. Biological applications at LLNL will be discussed.

Weber, P K; Fallon, S J; Pett-Ridge, J; Ghosal, S; Hutcheon, I D



Raman spectroscopic investigation of polycrystalline structures of CVD-grown graphene by isotope labeling.  


Topological defects, such as point defects, dislocations and grain boundaries, have a dramatic influence on the chemical and physical properties of large-scale graphene grown by chemical vapor deposition (CVD) method. Here we demonstrate the Raman visualization of polycrystalline structures in an isotopically modified CVD graphene. By means of the reversible reaction of methane on a copper catalyst, the etching of (12)C-lattice and surface deposition of (13)C-atoms occur in CVD graphene by sequentially introducing hydrogen and isotopic methane after standard growth of graphene with full monolayer coverage. Spatial Raman spectroscopic mapping on labeled graphene reveals pronounced network-like (13)C-rich regions, which are further identified to exist along the grain boundaries of graphene by low-energy electron microscopy. The structural defects inside the graphene grains are also targeted in the isotope labeling process. Our work opens a new way to investigate multiple grain structures in CVD graphene with a simple spectroscopic technique. PMID:25303722

Wang, Shengnan; Suzuki, Satoru; Hibino, Hiroki



Efficiency of background suppression for arterial spin labeling  

E-print Network

Arterial spin labeling (ASL), a technique developed for the measurement of local tissue perfusion with MRI, is heavily dependent on distinguishing irrelevant static tissue signal from the labeled blood. Background suppression ...

Garcia, Dairon, 1980-



Metabolism of selenite labelled with enriched stable isotope in the bloodstream.  


The metabolism of selenium (Se) in the bloodstream of rats was studied using HPLC-ICP-MS with an enriched Se stable isotope, and the results were used as Se-specific indicators for Se nutritional status. Concentration of endogenous Se in plasma depended on dietary Se, while changes in concentrations and distributions of exogenous Se revealed its metabolic pathway. Namely, selenite was taken up by red blood cells and reduced to selenide, and then reappeared in plasma in a form bound selectively to albumin within 10 min, disappeared from plasma again within 30 min after injection. Then, the concentration of labelled Se started to increase slowly as selenoprotein P and extracellular glutathione peroxidase, and attained a maximum level at about 6 h after injection. The isotope ratio of endogenous to exogenous Se concentrations in plasma after 48 h post-injection was proposed to represent the Se-specific indicator in plasma reflecting the nutritional status of Se. PMID:9187378

Suzuki, K T; Itoh, M



Stable Carbon Isotopes As Indicators of Plant Water Use Efficiency  

Microsoft Academic Search

Stable carbon isotopes have been utilized to better understand how environmental variables influence the efficiency of photosynthesis, specifically what factors limit the uptake and absorption of CO2 during photosynthesis. An understanding of the controls over both gas exchange and stomatal conductance can provide an explanation for the possible environmental influences on plant WUE. The delta13C of extractive-free wood was used

E. M. Powers; J. D. Marshall; N. Ubierna Lopez



Multiplexed analysis of cage and cage free chicken egg fatty acids using stable isotope labeling and mass spectrometry.  


Binary stable isotope labeling couple with LC-ESI-MS has been used as a powerful non-targeted approach for the relative quantification of lipids, amino acids, and many other important metabolite classes. A multiplexed approach using three or more isotopic labeling reagents greatly reduces analytical run-time while maintaining excellent sensitivity and reproducibility. Three isotopic cholamine labeling reagents have been developed to take advantage of the pre-ionized character of cholamine, for ESI, and the ease by which stable isotopes can be incorporated into the cholamine structure. These three cholamine labeling reagents have been used to relatively quantify three fatty acid samples simultaneously. The quantification resulted in the observation of 12 fatty acids that had an average absolute error of 0.9% and an average coefficient of variation of 6.1%. Caged versus cage-free isotope labeling experiments showed that cage-free eggs have an increased level of omega-3 fatty acids as compared to caged eggs. This multiplexed fatty acid analysis provides an inexpensive and expedited tool for broad-based lipid profiling that will further aid discoveries in the mechanisms of fatty acid action in cells. PMID:24317525

Torde, Richard G; Therrien, Andrew J; Shortreed, Michael R; Smith, Lloyd M; Lamos, Shane M



Multiplexed Analysis of Cage and Cage Free Chicken Egg Fatty Acids Using Stable Isotope Labeling and Mass Spectrometry  

PubMed Central

Binary stable isotope labeling couple with LC-ESI-MS has been used as a powerful non-targeted approach for the relative quantification of lipids, amino acids, and many other important metabolite classes. A multiplexed approach using three or more isotopic labeling reagents greatly reduces analytical run-time while maintaining excellent sensitivity and reproducibility. Three isotopic cholamine labeling reagents have been developed to take advantage of the pre-ionized character of cholamine, for ESI, and the ease by which stable isotopes can be incorporated into the cholamine structure. These three cholamine labeling reagents have been used to relatively quantify three fatty acid samples simultaneously. The quantification resulted in the observation of 12 fatty acids that had an average absolute error of 0.9% and an average coefficient of variation of 6.1%. Caged versus cage-free isotope labeling experiments showed that cage-free eggs have an increased level of omega-3 fatty acids as compared to caged eggs. This multiplexed fatty acid analysis provides an inexpensive and expedited tool for broad-based lipid profiling that will further aid discoveries in the mechanisms of fatty acid action in cells. PMID:24317525

Torde, Richard G.; Therrien, Andrew J.; Shortreed, Michael R.; Smith, Lloyd M.; Lamos, Shane M.



Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI  

PubMed Central

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs. PMID:23468856

D. M., Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen



Determination of protein conformation by isotopically labelled cross-linking and dedicated software  

NASA Astrophysics Data System (ADS)

Chemical cross-linking in conjunction with mass spectrometry (MS) can be used for sensitive and rapid investigation of the three-dimensional structure of proteins at low resolution. However, the resulting data are very complex, and on the bioinformatic side, there still exists an urgent need for improving computer software for (semi-) automated cross-linking data analysis. In this study, we have developed dedicated software for rapid and confident identification and validation of cross-linked species using an isotopic labelled cross-linker approach in combination with MS. Deuterated (+4 Da) and non-deuterated (+0 Da) bis(sulfosuccinimidyl)suberate, BS3, was used as homobifunctional cross-linker to tag the cross-linked regions. Peptides generated from proteolysis were separated using high performance liquid chromatography, and peptide mass fingerprinting was obtained for the individual fractions using matrix-assisted laser-desorption ionisation time-of-flight (MALDI TOF) MS. The resulting peptide mass lists were combined and transferred to the program, ProteinXXX, which generated the theoretical mass values of all combinations of cross-linked peptides and dead-end cross-links and compared this to the obtained mass lists. In addition, screening for 4 Da-separated signals aided the identification of potential cross-linked species. Sequence information of these candidates was then obtained using MALDI TOF TOF. The cross-linked peptides could then be validated based on the match of the fragmentation pattern and the theoretical values produced by ProteinXXX. This semi-automated interpretation provided a high analysis speed of cross-linking data, with efficient and confident identification of cross-linked species. Four experiments using different conditions showed a high degree of reproducibility as only 1 and 2 cross-links out of 36 identified was not observed in two experiments. The method was tested using human placenta calreticulin (CRT). Based on the identified cross-links, a few corrections to a model of calreticulin obtained by homology modelling using calnexin as template can be suggested. Furthermore, the cross-links show that the C-terminal of the protein continues along the core region opposite the P-domain for at least 11 residues beyond the known structure. In addition, it was observed that the conformation of CRT does not change significantly in the presence or absence of the divalent ions, Ca2+ and Zn2+.

Nielsen, Tina; Thaysen-Andersen, Morten; Larsen, Nanna; Jørgensen, Flemming S.; Houen, Gunnar; Højrup, Peter



Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.  


Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria. PMID:21959240

Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M



In vivo investigation of homocysteine metabolism to polyamines by high-resolution accurate mass spectrometry and stable isotope labeling.  


Polyamines are essential polycations, playing important roles in mammalian physiology. Theoretically, the involvement of homocysteine in polyamine synthesis via S-adenosylmethionine is possible; however, to our knowledge, it has not been established experimentally. Here, we propose an original approach for investigation of homocysteine metabolites in an animal model. The method is based on the combination of isotope-labeled homocysteine supplementation and high-resolution accurate mass spectrometry analysis. Structural identity of the isotope-labeled metabolites was confirmed by accurate mass measurements of molecular and fragment ions and comparison of the retention times and tandem mass spectrometry fragmentation patterns. Isotope-labeled methionine, spermidine, and spermine were detected in all investigated plasma and tissue samples. The induction of moderate hyperhomocysteinemia leads to an alteration in polyamine levels in a different manner. The involvement of homocysteine in polyamine synthesis and modulation of polyamine levels could contribute to a better understanding of the mechanisms connected with homocysteine toxicity. PMID:24736325

Ruseva, Silviya; Lozanov, Valentin; Markova, Petia; Girchev, Radoslav; Mitev, Vanio



Stable Carbon Isotopes As Indicators of Plant Water Use Efficiency  

NASA Astrophysics Data System (ADS)

Stable carbon isotopes have been utilized to better understand how environmental variables influence the efficiency of photosynthesis, specifically what factors limit the uptake and absorption of CO2 during photosynthesis. An understanding of the controls over both gas exchange and stomatal conductance can provide an explanation for the possible environmental influences on plant WUE. The ?13C of extractive-free wood was used as an index of plant water use efficiency at Mica Creek Experimental Watershed, Shoshone County, ID. The ?13C values of tree rings were used to determine the effects of clear cut and partial cut harvesting practices, the effect of elevation, and species differences in intrinsic water use efficiency (WUE) among coniferous species including: Thuja plicata, Larix occidentalis, Picea engelmannii, Pseudotsuga menziesii, Abies lasiocarpa, and Abies grandis. We found significant effects of harvest treatments (p=0.0197), elevation (p= 0.0268), and species (p<0.001) on tree ?13C. The significantly more enriched isotopic signatures observed in Thuja plicata (?13C = -23.37 ±0.17‰), indicate that it is a more water use efficient species compared to Larix occidentalis (?13C = -25.66 ±0.43‰), and Abies grandis (?13C = -25.83 ±0.15‰). There was also an overall trend of ?13C enrichment with elevation. The isotopic composition of tree rings has been estimated to increase by 0.003 ‰ per meter of elevation gain, which may be related to a decrease in soil moisture with elevation. Finally, the mean ?13C values observed on partial cut (?13C = -24.73 ±0.10‰) and clear cut treatments (?13C = -24.45 ±0.29‰) were significantly more enriched than the mean value for the control treatment (?13C = -25.25 ±0.19‰). The more enriched isotopic signatures observed on the harvested treatments indicate that the trees are more water use efficient, which may be a result of increased photosynthetic capacity with an increase in the availability of water, foliar nitrogen, and light to individual trees on the harvested treatments. The reduction of stand density through harvesting may reduce the transpirational water losses on a stand level, thus increasing the water availability for individual trees.

Powers, E. M.; Marshall, J. D.; Ubierna Lopez, N.



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

NASA Astrophysics Data System (ADS)

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 analogs ( 2H 12- and/or 2H 12- 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 O 2 concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O 2 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.

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



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

PubMed Central

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

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



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


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 analogs ((2)H(12)- and/or (2)H(12)-(15)N-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 O(2) concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O(2) sensitivity. Labeling the nitroxides with (15)N 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

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



Application of stable isotope labelling in cell culture experiments: [2-13 C]pyruvate as novel and superior substrate for in vitro  

E-print Network

Application of stable isotope labelling in cell culture experiments: [2-13 C]pyruvate as novel, Germany Introduction: Use of stable isotope labeling in cell culture experiments is a widely applied potassium hydroxide solution and lyophilized. NMR analysis: Lyophilized samples were redissolved in 0.5 ml D


Cost-effective production of 13C, 15N stable isotope-labelled biomass from phototrophic microalgae for various biotechnological applications.  


The present study outlines a process for the cost-effective production of 13C/15N-labelled biomass of microalgae on a commercial scale. The core of the process is a bubble column photobioreactor with exhaust gas recirculation by means of a low-pressure compressor. To avoid accumulation of dissolved oxygen in the culture, the exhaust gas is bubbled through a sodium sulphite solution prior to its return to the reactor. The engineered system can be used for the production of 13C, 15N, and 13C-15N stable isotope-labelled biomass as required. To produce 13C-labelled biomass, 13CO2 is injected on demand for pH control and carbon supply, whereas for 15N-labelled biomass Na15NO3 is supplied as nitrogen source at the stochiometric concentration. The reactor is operated in semicontinuous mode at different biomass concentrations, yielding a maximum mean biomass productivity of 0.3 gL(-1) day(-1). In order to maximize the uptake efficiency of the labelled substrates, the inorganic carbon is recovered from the supernatant by acidification/desorption processes, while the nitrate is delivered at stochiometric concentration and the harvesting of biomass is performed when the 15NO3- is depleted. In these conditions, elemental analysis of both biomass and supernatant shows that 89.2% of the injected carbon is assimilated into the biomass and 6.9% remains in the supernatant. Based on elemental analysis, 97.8% of the supplied nitrogen is assimilated into the biomass and 1.3% remains in the supernatant. Stable isotope-labelling enrichment has been analysed by GC-MS results showing that the biomass is highly labelled. All the fatty acids are labelled; more than 96% of the carbon present in these fatty acids is 13C. The engineered system was stably operated for 3 months, producing over 160 g of 13C and/or 15N-labelled biomass. The engineered bioreactor can be applied for the labelling of various microalgae. PMID:16257578

Acién Fernández, F G; Fernández Sevilla, J M; Egorova-Zachernyuk, T A; Molina Grima, E



Negative ion ESI-MS analysis of natural yellow dye flavonoids--An isotopic labelling study  

NASA Astrophysics Data System (ADS)

Flavonoids are amongst the most commonly used natural yellow colourants in paintings, as lakes, and in historical textiles as mordant dyes. In this paper, evidence from isotopically labelled substrates is used to propose negative ion electrospray collision induced decomposition mechanisms of flavones, flavonols and an isoflavone. These mechanisms include a retro-Diels-Alder fragmentation (observed for flavones and flavonols) and an M-122 fragmentation (characteristic of 3',4'-dihydroxyflavonols). In addition, the presence of a m/z 125 fragment ion is shown to be characteristic of 2'-hydroxyflavonols and an ion at m/z 149 is shown to be characteristic of 4'-hydroxyflavones. Applications of these methods are exemplified by the identification of a minor component of Dyer's camomile (Anthemis tinctoria L.) and the identification of the dye source in green threads sampled from an 18th Century Scottish tartan fragment.

McNab, Hamish; Ferreira, Ester S. B.; Hulme, Alison N.; Quye, Anita



Ultrafast, Unimpeded Liquid Water Transport Through Graphene-Based Nanochannels Measured by Isotope Labelling  

E-print Network

Graphene-based laminates, with ultralong and tortuous nanocapillaries formed by simply stacking graphene flakes together, have great promises in filtration and separation. However, the information on liquid water trans-membrane permeation is lacking, which is the most fundamental problem and of crucial importance in solution-based mass transport. Here, based on isotope labelling, we investigate the liquid water transportation through graphene-based nanocapillaries under no external hydrostatic pressures. Liquid water can afford an unimpeded permeation through graphene-based nanochannels with a diffusion coefficient 4~5 orders of magnitude larger than through sub-micrometer-sized polymeric channels. When dissolving ions in sources, the diffusion coefficient of ions through graphene channels lies in the same order of magnitude as water, while the ion diffusion is faster than water, indicating that the ions are mainly transported by fast water flows and the delicate interactions between ions and nanocapillary wa...

Sun, Pengzhan; Wang, Kunlin; Zhong, Minlin; Wu, Dehai; Zhu, Hongwei



Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling.  


Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans. PMID:17137339

Atwood, James A; Minning, Todd; Ludolf, Fernanda; Nuccio, Arthur; Weatherly, Daniel B; Alvarez-Manilla, Gerardo; Tarleton, Rick; Orlando, Ron



Oxygen Atom Transfer and Oxidative Water Incorporation in Cuboidal Mn3MOn Complexes Based on Synthetic, Isotopic Labeling,  

E-print Network

, United States *S Supporting Information ABSTRACT: The oxygen-evolving complex (OEC) of photo- system II is performed by the oxygen-evolving center (OEC) of photo- system II (PSII).2 The OEC consists of a Mn4Ca on Synthetic, Isotopic Labeling, and Computational Studies Jacob S. Kanady,§, Jose L. Mendoza-Cortes,, Emily Y

Goddard III, William A.


Quantification of protein deposits on silicone hydrogel materials using stable-isotopic labeling and multiple reaction monitoring  

Microsoft Academic Search

This study was designed to use multiple reaction monitoring (MRM) for accurate quantification of contact lens protein deposits. Worn lenses used with a multipurpose disinfecting solution were collected after wear. Individual contact lenses were extracted and then digested with trypsin. MRM in conjunction with stable-isotope-labeled peptide standards was used for protein quantification. The results show that lysozyme was the major

Negar Babaei Omali; Zhenjun Zhao; Ling Zhong; Mark J. Raftery; Hua Zhu; Jerome Ozkan; Mark Willcox



Carbon isotope labeling in boreal forests to assess roles of fungal species in decomposition  

NASA Astrophysics Data System (ADS)

We used 14C and 13C labeling to assess the in situ respiration of alanine-, starch-, and lignocellulose-derived carbon from the sporocarps of particular fungal species fruiting in a boreal forest in Alaska. By measuring isotopically-labeled respiration of sporocarps, which can be identified to species, we were able to attribute turnover of carbon compounds to specific fungal groups. Moreover, collection of sporocarp respiration is non-destructive, so we could return to the same sporocarps to collect a time series of measurements that spanned hours to days. We tested the hypotheses that alanine and starch turn over more quickly than lignocellulose, and that saprotrophic fungi would use starch-C and lignocellulose-C but ectomycorrhizal fungi would not. Small amounts of 14C-labeled alanine (about 100,000 permil) were dispensed into the soil within three meters of sporocarps of the ectomycorrhizal fungus Lactarius alnicola. ?14CO2 values of sporocarp respiration climbed from 75.8 +/- 6.3 permil to 7855 +/- 3940 permil within one hour of additions, indicating that the fungus quickly acquired, transported, and transformed the alanine-C. In a separate approach, a mixture of 13C-labeled starch (about 15,000 permil) and 14C-labeled lignocellulose (about 36,000 permil) was applied in 9 m2 plots containing sporocarps of the ectomycorrhizal genera Phellodon and Sarcodon and the saprotrophic genera Lycoperdon and Polyporus. An unlabeled control plot was also established. We observed no detectable increase in 14CO2 or 13CO2 over a 144 hour period, suggesting that neither ectomycorrhizal nor saprotrophic fungi significantly broke down starch or lignocellulose during this time. The alanine experiment is one of the first to indicate that ectomycorrhizal fungi can influence the spatial distribution and storage of soil carbon over short time scales. This influence may be restricted to carbon of organic compounds like amino acids. In contrast, starch was not transformed quickly even by saprotrophic fungi, which may be due to an absence or lack of activity of starch-degrading fungal species during the study period. Potential activity of the starch-metabolizing enzyme alpha-glucosidase was only 0.59 +/- 0.17 ìmol h-1 g dry soil-1, which was 7 times less than activity of beta-glucosidase, which breaks down cellulose. The slow turnover of lignocellulose-C was consistent with slow decomposition rates of plant litter in this biome.

Treseder, K. K.; Czimczik, C. I.; Trumbore, S. E.; Allison, S. D.



Whole proteome analysis of the protozoan parasite Trypanosoma brucei using stable isotope labeling by amino acids in cell culture and mass spectrometry.  


The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time has allowed for the characterization of the proteome from several different life stages of the parasite (Butter et al., Mol Cell Proteomics 12:172-179, 2013; Gunasekera et al., BMC Genomics 13:556, 2012; Urbaniak et al., PloS One 7(5):e36619, 2012). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC) (Ong et al., Mol Cell Proteomics 1:376-386, 2002) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knockout approaches. PMID:25059603

Cirovic, Olivera; Ochsenreiter, Torsten



Location of Structural Transitions in an Isotopically Labeled Lung Surfactant SP-B Peptide by IRRAS  

PubMed Central

Pulmonary surfactant, a lipid/protein complex that lines the air/water interface in the mammalian lung, functions to reduce the work of breathing. Surfactant protein B (SP-B) is a small, hydrophobic protein that is an essential component of this mixture. Structure-function relationships of SP-B are currently under investigation as the protein and its peptide analogs are being incorporated into surfactant replacement therapies. Knowledge of the structure of SP-B and its related peptides in bulk and monolayer phases will facilitate the design of later generation therapeutic agents. Prior infrared reflection-absorption spectroscopic studies reported notable, reversible surface pressure-induced antiparallel ?-sheet formation in a synthetic peptide derived from human SP-B, residues 9–36 (SP-B9–36). In the current work, infrared reflection-absorption spectroscopy is applied in conjunction with isotopic labeling to detect the site and pressure dependence of the conformational change. SP-B9–36, synthesized with 13C=O-labeled Ala residues in positions 26, 28, 30, and 32, shifted the ?-sheet marker band to ?1600 cm?1 and thus immediately identified this structural element within the labeled region. Surface pressure-induced alterations in the relative intensities of Amide I band constituents are interpreted using a semiempirical transition dipole coupling model. In addition, electron micrographs reveal the formation of tubular myelin structures from in vitro preparations using SP-B9–36 in place of porcine SP-B indicating that the peptide has the potential to mimic this property of the native protein. PMID:12829488

Flach, Carol R.; Cai, Peng; Dieudonné, Darline; Brauner, Joseph W.; Keough, Kevin M. W.; Stewart, June; Mendelsohn, Richard



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

NASA Astrophysics Data System (ADS)

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

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



A Method for Sporulating Budding Yeast Cells That Allows for Unbiased Identification of Kinase Substrates Using Stable Isotope Labeling by Amino Acids in Cell Culture  

PubMed Central

Quantitative proteomics has been widely used to elucidate many cellular processes. In particular, stable isotope labeling by amino acids in cell culture (SILAC) has been instrumental in improving the quality of data generated from quantitative high-throughput proteomic studies. SILAC uses the cell’s natural metabolic pathways to label proteins with isotopically heavy amino acids. Incorporation of these heavy amino acids effectively labels a cell’s proteome, allowing the comparison of cell cultures treated under different conditions. SILAC has been successfully applied to a variety of model organisms including yeast, fruit flies, plants, and mice to look for kinase substrates as well as protein–protein interactions. In budding yeast, several kinases are known to play critical roles in different aspects of meiosis. Therefore, the use of SILAC to identify potential kinase substrates would be helpful in the understanding the specific mechanisms by which these kinases act. Previously, it has not been possible to use SILAC to quantitatively study the phosphoproteome of meiotic Saccharomyces cerevisiae cells, because yeast cells sporulate inefficiently after pregrowth in standard synthetic medium. In this study we report the development of a synthetic, SILAC-compatible, pre-sporulation medium (RPS) that allows for efficient sporulation of S. cerevisiae SK1 diploids. Pre-growth in RPS supplemented with heavy amino acids efficiently labels the proteome, after which cells proceed relatively synchronously through meiosis, producing highly viable spores. As proof of principle, SILAC experiments were able to identify known targets of the meiosis-specific kinase Mek1. PMID:25168012

Suhandynata, Ray; Liang, Jason; Albuquerque, Claudio. P.; Zhou, Huilin; Hollingsworth, Nancy M.



Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins  

E-print Network

Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with mutant forms of MetRS. Link and coworkers recently reported a high-throughput screen for E. coli Met

Goddard III, William A.


Experimental investigation of rates and mechanisms of isotope exchange (O, H) between volcanic ash and isotopically-labeled water  

NASA Astrophysics Data System (ADS)

The hydrogen and oxygen isotope ratios in hydrous minerals and volcanic glass are routinely used as paleo-proxies to infer the isotopic values of meteoric waters and thus paleo-climatic conditions. We report a series of long-term exposure experiments of distal 7700 BP Mt. Mazama ash (-149‰ ?2H, +7‰ ?18O, 3.8 wt.% H2O) with isotopically-labeled water (+650‰ ?2H, +56‰ ?18O). Experiments were done at 70, 40 and 20 °C, and ranged in duration from 1 to 14454 h (˜20 months), to evaluate the rates of deuterium and 18O exchange, and investigate the relative role of exchange and diffusion. We also investigate the effect of drying on H2Otot and ?2H in native and reacted ash that can be used in defining the protocols for natural sample preparation. We employ Thermal Conversion Elemental Analyzer (TCEA) mass spectrometry, thermogravimetric analysis and a KBr pellet technique with infrared spectroscopy to measure the evolution of ?2H, total water, and OH water peaks in the course of exposure experiments, and in varying lengths of vacuum drying. Time series experiments aided by infrared measurements demonstrate the following new results: (i) It wasobserved that from 5 to >100‰ ?2H increases with time, with faster deuterium exchange at higher temperatures. Times at 15% of theoretical "full ?2H exchange" are: 15.8 years at 20 °C, 5.2 years at 40 °C, and 0.4 years at 70 °C. (ii) Even at extended exposure durations experiments show no net increase in water weight percent nor in ?18O in ash; water released from ash rapidly by thermal decomposition is not enriched in ?18O. This observation clearly suggests that it is hydrogen exchange, and not water addition or oxygen exchange that characterizes the process. (iii) Our time series drying, Fourier transform infrared (FTIR)-KBr and Thermogravimetric Analyzer (TGA) analyses collectively suggest a simple mechanistic view that there are three kinds of "water" in ash: water (mostly H2O) that is less strongly bonded on the surface of ash particles that are getting lost with 24-48 h of drying to up to 200-300 °C, bound water in glass in a range of combining proportions of SiOH to H2O, and a small reservoir of residual, tightly held water. Experimentation with vacuum drying at 130-220 °C, and with TGA up to 1000 °C provides a set of simple relationships and recommendations for users. Ash loses 1-1.2 wt.% water with weight loss stabilizing after 48 h of vacuum drying at 130 °C. This ash drying removes molecular water over the hydroxyl group in a proportion of ˜80:20% resulting in relatively constant ?2H values of the remaining total water in native ash. This study demonstrates that ?2H in ash can be rapidly changed by minor diagenesis even at relatively low temperatures of 20 °C. A diagenetic history of ash is needed to interpret the D/H ratio, but the ?18O values of water in ash are more robust.

Nolan, Gary S.; Bindeman, Ilya N.



Investigation of the mechanism of n-butane oxidation on vanadium phosphorus oxide catalysts: evidence from isotopic labeling studies.  


The selective oxidation of n-butane to maleic acid catalyzed by vanadium phosphates (VPO) is one of the most complex partial oxidation reactions used in industry today. Numerous reaction mechanisms have been proposed in the literature, many of which have butenes, butadiene, and furan as reaction intermediates. We have developed an experimental protocol to study the mechanism of this reaction in which (13)C-isotopically labeled n-butane is flowed over a catalyst bed and the reaction products are analyzed using (13)C NMR spectroscopy. This protocol approximates the conditions found in an industrial reactor without requiring an exorbitant amount of isotopically labeled material. When [1,4-(13)C]n-butane reacted on VPO catalysts to produce maleic acid and butadiene, the isotopic labels were observed in both the 1,4 and 2,3 positions of butadiene and maleic acid. The ratio of label scrambling was typically 1:20 for the 2,3:1,4 positions in maleic acid. For butadiene, the ratio of label scrambling was consistently much higher, at 2:3 for the 2,3:1,4 positions. Because of the discrepancy in the amount of label scrambling between maleic acid and butadiene, butadiene is unlikely to be the primary reaction intermediate for the conversion of n-butane to maleic anhydride under typical industrial conditions. Ethylene was always observed as a side product for n-butane oxidation on VPO catalysts. Fully (13)C-labeled butane produced about 5-13 times as much isotopically labeled ethylene as did [1,4-(13)C]butane, indicating that ethylene was produced mainly from the two methylene carbons of n-butane. When the reaction was run under conditions which minimize total oxidation products such as CO and CO(2), the amounts of ethylene and carbon oxides produced from fully (13)C-labeled butane were almost equal. This strongly suggests that the total oxidation of n-butane on VPO catalysts involves the oxidation and abstraction of the two methyl groups of n-butane, and the two methylene groups of n-butane form ethylene. An organometallic mechanism is proposed to explain these results. PMID:11853438

Chen, Bin; Munson, Eric J



Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly  

PubMed Central

In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (13C6-glucose) for 12 h – either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate – possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism. PMID:23185587

Tseng, Te-Wei; Wu, June-Tai; Chen, Yu-Chie; Urban, Pawel L.



Measuring supply chain carbon efficiency : a carbon label framework  

E-print Network

In the near term, efficiency improvements represent a key option for reducing the impacts of climate change. The growing awareness of climate change has increased the attention regarding the carbon emissions "embedded" in ...

Craig, Anthony (Anthony J.)



Urinary metabolites of 2-bromoethanamine identified by stable isotope labelling: evidence for carbamoylation and glutathione conjugation.  


2-Bromoethanamine (BEA) causes renal papillary necrosis (RPN) in rats after a single dose and has been widely used as a model compound for studying the lesion. Although the metabolism of BEA may be an important determinant of toxicity, the metabolic fate of the compound has not been fully elucidated. To date, the only identified BEA metabolites are aziridine, 2-oxazolidone and 5-hydroxy-2-oxazolidone. In this study, stable isotope labelling (SIL) of BEA analogs ((¹³C and ²H) were used to differentiate generated BEA metabolites from endogenous molecules which enabled the accurate liquid chromatography mass spectrometry detection of more than 180 novel metabolites. BEA metabolism was evaluated in rats after acute administration of a non-toxic dose (50 mg/kg) and a toxic dose (250 mg/kg) that caused frank RPN and polyuria. Newly identified metabolites include three carbamoylation products, two mercapturic acids and a group of amino acid conjugates. Overall, the results indicate that BEA metabolism is very complex, suggest the potential formation of reactive intermediates and establish that BEA is subject to conjugation with glutathione. The results also demonstrate the utility and sensitivity of the SIL approach for identification of metabolites from small, reactive compounds. PMID:21043805

Shipkova, Petia; Vassallo, Jeffrey D; Aranibar, Nelly; Hnatyshyn, Serhiy; Zhang, Haiying; Clayton, T Andrew; Cantor, Glenn H; Sanders, Mark; Coen, Muireann; Lindon, John C; Holmes, Elaine; Nicholson, Jeremy K; Lehman-McKeeman, Lois



LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma  

NASA Technical Reports Server (NTRS)

Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.

Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi



Investigation of bn-44 Peptide Fragments Using High Resolution Mass Spectrometry and Isotope Labeling.  


An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic bn-44 ions instead of bn-28 (an) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH3CHO (44.0262) from a Thr side chain; (2) loss of CO2 (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel bn-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway. PMID:25280401

Wang, Bing; Yu, Jiayi; Wang, Huixin; Wei, Zhonglin; Guo, Xinhua; Xiao, Zhaohui; Zeng, Zhoufang; Kong, Wei



Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS  

NASA Astrophysics Data System (ADS)

N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

Zhu, Zhikai; Go, Eden P.; Desaire, Heather



Isotope labeling-based quantitative proteomics of developing seeds of castor oil seed (Ricinus communis L.).  


In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism, seed-storage proteins (SSPs), toxins, and allergens. Additionally, we have used off-line hydrophilic interaction chromatography (HILIC) as a step of peptide fractionation preceding the reverse-phase nanoLC coupled to a LTQ Orbitrap. We were able to identify a total of 1875 proteins, and from these 1748 could be mapped to extant castor gene models, considerably expanding the number of proteins so far identified from developing castor seeds. Cluster validation and statistical analysis resulted in 975 protein trend patterns and the relative abundance of 618 proteins. The results presented in this work give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism, and catabolism of fatty acid and the pattern of deposition of SSPs, toxins, and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of SSP that are differentially expressed during seed development. PMID:24090105

Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit; Soares, Emanuela L; Soares, Arlete A; Roepstorff, Peter; Domont, Gilberto B; Campos, Francisco A P



A device for single leaf labelling with CO2 isotopes to study carbon allocation and partitioning in Arabidopsis thaliana  

PubMed Central

Background Plant biomass consists primarily of carbohydrates derived from photosynthesis. Monitoring the assimilation of carbon via the Calvin-Benson cycle and its subsequent utilisation is fundamental to understanding plant growth. The use of stable and radioactive carbon isotopes, supplied to plants as CO2, allows the measurement of fluxes through the intermediates of primary photosynthetic metabolism, long-distance transport of sugars in the vasculature, and the synthesis of structural and storage components. Results Here we describe the design of a system for supplying isotopically labelled CO2 to single leaves of Arabidopsis thaliana. We demonstrate that the system works well using short pulses of 14CO2 and that it can be used to produce robust qualitative and quantitative data about carbon export from source leaves to the sink tissues, such as the developing leaves and the roots. Time course experiments show the dynamics of carbon partitioning between storage as starch, local production of biomass, and export of carbon to sink tissues. Conclusion This isotope labelling method is relatively simple to establish and inexpensive to perform. Our use of 14CO2 helps establish the temporal and spatial allocation of assimilated carbon during plant growth, delivering data complementary to those obtained in recent studies using 13CO2 and MS-based metabolomics techniques. However, we emphasise that this labelling device could also be used effectively in combination with 13CO2 and MS-based techniques. PMID:24252607



Molecularly imprinted solid phase extraction using stable isotope labeled compounds as template and liquid chromatography–mass spectrometry for trace analysis of bisphenol A in water sample  

Microsoft Academic Search

We have developed a molecularly imprinted polymer (MIP) using a stable isotope labeled compound as the template molecule and called it the “isotope molecularly imprinted polymer” (IMIP). In this study, bisphenol A (BPA) was used as the model compound. None imprinted polymer (NIP), MIP, dummy molecularly imprinted polymer (DMIP) and IMIP were prepared by the suspension polymerization method using without

Migaku Kawaguchi; Yoshio Hayatsu; Hisao Nakata; Yumiko Ishii; Rie Ito; Koichi Saito; Hiroyuki Nakazawa



Binding of isotopically labeled substrates, inhibitors, and cyanide by protocatechuate 3,4-dioxygenase  

SciTech Connect

Binding of ligands to the active site Fe3+ of protocatechuate 3,4-dioxygenase is investigated using EPR-detected transferred hyperfine coupling from isotopically labeled substrates, inhibitors, and cyanide. Broadening is observed in EPR resonances from the anaerobic enzyme complex with homoprotocatechuate (3,4-dihydroxyphenylacetate), a slow substrate, enriched with 17O (I = 5/2) in either the 3-OH or the 4-OH group. This shows that this substrate binds directly to the Fe3+ and strongly suggests that an iron chelate can be formed. Cyanide is known to bind to the enzyme in at least two steps, forming first a high spin and then a low spin complex. Hyperfine broadening from (13C)cyanide (I = 1/2) is observed in the EPR spectra of both complexes, showing that cyanide is an Fe3+ ligand in each case. Cyanide binding is also at least biphasic in the presence of protocatechuate (PCA). The initial high spin enzyme-PCA-cyanide complex forms rapidly and exhibits a unique EPR spectrum. Broadening from PCA enriched with 17O in either the 3-OH or the 4-OH group is detected showing that PCA binds to the iron, probably as a chelate complex. In contrast, no broadening from (13C)cyanide is detected for this complex suggesting that cyanide binds at a site away from the Fe3+. Steady state kinetic measurements of cyanide inhibition of PCA turnover are consistent with two rapidly exchanging cyanide binding sites that inhibit PCA binding and which can be simultaneously occupied. Formation of the nearly irreversible, low spin enzyme-PCA-cyanide complex is competitively inhibited by PCA. Transient kinetics of the formation of this complex are second order in cyanide implying that two cyanides bind. Broadening in the EPR spectrum of this complex is detected from (13C)cyanide, but not from (17O)PCA, suggesting that PCA is displaced.

Orville, A.M.; Lipscomb, J.D.



Evaluating SPIO-labelled cell MR efficiency by three-dimensional quantitative T2* MRI.  


An in vitro MR-assay for superparamagnetic iron oxide (SPIO) particle cell labelling assessment via three-dimensional quantitative T(2) (*) MR microscopy was proposed. On high-resolution images, and due to the high susceptibility difference between the particles and the surrounding medium, SPIO internalized in cells induces signal loss which may be counted and measured on T(2) (*) maps. The increase in both labelled cell percentage and the average perturbation volume with an added amount of iron in the incubation medium proved that intracellular iron uptake is dependent upon the initial concentration of incubation iron. It also proved that the observed increases in total cellular iron uptake measured by inductively coupled plasma optical emission spectroscopy are due to both an increase in the iron mass per cell and also an increase in labelled cell concentration. MR results were compared with Prussian blue staining histology. The sensitivity of the MR methodology was then used to distinguish labelling differences for two different types of particle coating. The MRI-assay we proposed is a compulsory tool to optimize labelling efficiency in order to improve in vivo cell detection. Key parameters for detection, such as the percentage of cell labelling, the effect on the image for a given amount of internalized iron and labelling distribution among a cell population, are easily obtained. The comparison of different contrast agents for labelling one cell type, the assessment of one type of contrast agent for labelling different cell types and/or the evaluation of labelling strategies, are possible without having recourse to classical methods, and provide improved accuracy, since the principle is based on intracellular relaxivity. PMID:16998951

Mowat, P; Franconi, F; Chapon, C; Lemaire, L; Dorat, J; Hindré, F; Benoit, J-P; Richomme, P; Le Jeune, J-J



An efficient technique for primer development and application that integrates fluorescent labeling and multiplex PCR1  

PubMed Central

• Premise of the study: Development of genetic markers can be costly and time-consuming, especially when multiple primer pairs are fluorescently labeled. This step was streamlined by combining two techniques in the same PCR reaction: (1) custom-labeling of primers by the investigator and (2) multiplexing multiple primers together in the same reaction. • Methods and Results: This technique was successfully used to develop microsatellite markers in several plant species. Microsatellites amplified with this multiplexing process were identical to those generated from PCR using individual primer pairs and with traditional methods using a priori labeled fluorescent primers. Tests of PCR cycling programs revealed that conditions recommended for the commercial kit generated stronger fragment peaks than the previously recommended cycling protocol. • Conclusions: This technique is an efficient and economical way to fluorescently label multiple microsatellite primers in the same reaction. It is also applicable to other markers used in PCR amplification of genetic material. PMID:25202486

Culley, Theresa M.; Stamper, Trevor I.; Stokes, Richard L.; Brzyski, Jessica R.; Hardiman, Nicole A.; Klooster, Matthew R.; Merritt, Benjamin J.



Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A Novel Glycan-Relative Quantification Strategy  

NASA Astrophysics Data System (ADS)

The Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT) strategy for the sample preparation, data analysis, and relative quantification of N-linked glycans is presented. Glycans are derivatized with either natural (L) or stable-isotope labeled (H) hydrazide reagents and analyzed using reversed phase liquid chromatography coupled online to a Q Exactive mass spectrometer. A simple glycan ladder, maltodextrin, is first used to demonstrate the relative quantification strategy in samples with negligible analytical and biological variability. It is shown that after a molecular weight correction attributable to isotopic overlap and a post-acquisition normalization of the data to account for any systematic bias, a plot of the experimental H:L ratio versus the calculated H:L ratio exhibits a correlation of unity for maltodextrin samples mixed in different ratios. We also demonstrate that the INLIGHT approach can quantify species over four orders of magnitude in ion abundance. The INLIGHT strategy is further demonstrated in pooled human plasma, where it is shown that the post-acquisition normalization is more effective than using a single spiked-in internal standard. Finally, changes in glycosylation are able to be detected in complex biological matrices, when spiked with a glycoprotein. The ability to spike in a glycoprotein and detect change at the glycan level validates both the sample preparation and data analysis strategy, making INLIGHT an invaluable relative quantification strategy for the field of glycomics.

Walker, S. Hunter; Taylor, Amber D.; Muddiman, David C.



Fast methodology for the reliable determination of nonylphenol in water samples by minimal labeling isotope dilution mass spectrometry.  


In this work we have developed and validated an accurate and fast methodology for the determination of 4-nonylphenol (technical mixture) in complex matrix water samples by UHPLC-ESI-MS/MS. The procedure is based on isotope dilution mass spectrometry (IDMS) in combination with isotope pattern deconvolution (IPD), which provides the concentration of the analyte directly from the spiked sample without requiring any methodological calibration graph. To avoid any possible isotopic effect during the analytical procedure the in-house synthesized (13)C1-4-(3,6-dimethyl-3-heptyl)phenol was used as labeled compound. This proposed surrogate was able to compensate the matrix effect even from wastewater samples. A SPE pre-concentration step together with exhaustive efforts to avoid contamination were included to reach the signal-to-noise ratio necessary to detect the endogenous concentrations present in environmental samples. Calculations were performed acquiring only three transitions, achieving limits of detection lower than 100ng/g for all water matrix assayed. Recoveries within 83-108% and coefficients of variation ranging from 1.5% to 9% were obtained. On the contrary a considerable overestimation was obtained with the most usual classical calibration procedure using 4-n-nonylphenol as internal standard, demonstrating the suitability of the minimal labeling approach. PMID:23746647

Fabregat-Cabello, Neus; Castillo, Ángel; Sancho, Juan V; González, Florenci V; Roig-Navarro, Antoni Francesc



Energy-efficiency labels and standards: A guidebook for appliances, equipment and lighting  

SciTech Connect

Energy-performance improvements in consumer products are an essential element in any government's portfolio of energy-efficiency and climate change mitigation programs. Governments need to develop balanced programs, both voluntary and regulatory, that remove cost-ineffective, energy-wasting products from the marketplace and stimulate the development of cost-effective, energy-efficient technology. Energy-efficiency labels and standards for appliances, equipment, and lighting products deserve to be among the first policy tools considered by a country's energy policy makers. The U.S. Agency for International Development (USAID) and the United Nations Foundation (UNF) recognize the need to support policy makers in their efforts to implement energy-efficiency standards and labeling programs and have developed this guidebook, together with the Collaborative Labeling and Appliance Standards Program (CLASP), as a primary reference. This guidebook was prepared over the course of the past year with significant contribution from the authors and reviewers mentioned previously. Their diligent participation has made this the international guidance tool it was intended to be. The lead authors would also like to thank the following individuals for their support in the development, production, and distribution of the guidebook: Marcy Beck, Elisa Derby, Diana Dhunke, Ted Gartner, and Julie Osborn of Lawrence Berkeley National Laboratory as well as Anthony Ma of Bevilacqua-Knight, Inc. This guidebook is designed as a manual for government officials and others around the world responsible for developing, implementing, enforcing, monitoring, and maintaining labeling and standards-setting programs. It discusses the pros and cons of adopting energy-efficiency labels and standards and describes the data, facilities, and institutional and human resources needed for these programs. It provides guidance on the design, development, implementation, maintenance, and evaluation of the programs and on the design of the labels and standards themselves. In addition, it directs the reader to references and other resources likely to be useful in conducting the activities described and includes a chapter on energy policies and programs that complement appliance efficiency labels and standards. This guidebook attempts to reflect the essential framework of labeling and standards programs. It is the intent of the authors and sponsors to distribute copies of this book worldwide at no charge for the general public benefit. The guidebook is also available on the web at and can be downloaded to be used intact or piecemeal for whatever beneficial purposes readers may conceive.

McMahon, James E.; Wiel, Stephen



Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napus Embryos1  

PubMed Central

Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mm carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mm amino acids as well as 6 to 15 mm malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing 13C-labeled carbohydrates. The 13C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of 13C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of 13C label into plastid-formed fatty acids, but substantially diluted 13C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. 13C label in the terminal acetate units of C20 and C22 fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute 13C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos. PMID:12226514

Schwender, Jorg; Ohlrogge, John B.



Efficient isotope separation by single-photon atomic sorting  

SciTech Connect

We propose a general and scalable approach to isotope separation. The method is based on an irreversible change of the mass-to-magnetic moment ratio of a particular isotope in an atomic beam, followed by a magnetic multipole whose gradients deflect and guide the atoms. The underlying mechanism is a reduction of the entropy of the beam by the information of a single scattered photon for each atom that is separated. We numerically simulate isotope separation for a range of examples, which demonstrate this technique's general applicability to almost the entire periodic table. The practical importance of the proposed method is that large-scale isotope separation should be possible, using ordinary inexpensive magnets and the existing technologies of supersonic beams and lasers.

Jerkins, M.; Chavez, I.; Raizen, M. G. [Center for Nonlinear Dynamics and Department of Physics, University of Texas at Austin, Austin, Texas 78712 (United States); Even, U. [Sackler School of Chemistry, Tel-Aviv University, Tel-Aviv (Israel)



VLDL apolipoprotein B-100, a potential indicator of the isotopic labeling of the hepatic protein synthetic precursor pool in humans: studies with multiple stable isotopically labeled amino acids.  


Four adult men received a 48-h constant intravenous infusion of [2H4]lysine, [2H3]leucine, L-[ring-13C6]phenylalanine, and L-[1,2,3,-13C3]alanine. Subjects ingested hourly meals for two 12-h periods, separated to two 12-h fasting periods. The isotopic enrichments of free amino acids in venous plasma and in VLDL apolipoprotein B-100 (apoB)-bound amino acids, plasma alpha-keto isocaproic acid (alpha-KIC) and plasma pyruvic acid (PYR) were measured by negative chemical ionization gas chromatography-mass spectrometry. By 7 h of infusion, all four amino acids achieved an equilibrium isotopic enrichment (EIE) in plasma and in apoB. In the fed state, the EIE of the amino acids in apoB was lower than that in plasma free amino acids. The ratio EIE-apoB:EIE-plasma differed significantly among amino acids in the fed state (alanine 0.30; lysine 0.64; leucine 0.70; phenylalanine 0.81). In the postabsorptive state, the EIE-apoB:EIE-plasma ratio rose significantly compared with the fed state (alanine 0.38; lysine 0.73; leucine 0.94; phenylalanine 1.05). Plasma PYR and apoB-alanine were in isotopic equilibrium irrespective of nutritional state. The EIE-apoB-leucine:EIE-plasma-alpha-KIC ratio rose from 0.75 in the fed state to near 1 in the postabsorptive state. We conclude that the contribution of systemic amino acids to apoB-100 synthesis is sensitive to nutritional state, and that systemic essential amino acids seem to be preferentially incorporated into apoB. PMID:1542004

Reeds, P J; Hachey, D L; Patterson, B W; Motil, K J; Klein, P D



CK-LPA: Efficient community detection algorithm based on label propagation with community kernel  

NASA Astrophysics Data System (ADS)

With the rapid development of Web 2.0 and the rise of online social networks, finding community structures from user data has become a hot topic in network analysis. Although research achievements are numerous at present, most of these achievements cannot be adopted in large-scale social networks because of heavy computation. Previous studies have shown that label propagation is an efficient means to detect communities in social networks and is easy to implement; however, some drawbacks, such as low accuracy, high randomness, and the formation of a “monster” community, have been found. In this study, we propose an efficient community detection method based on the label propagation algorithm (LPA) with community kernel (CK-LPA). We assign a corresponding weight to each node according to node importance in the whole network and update node labels in sequence based on weight. Then, we discuss the composition of weights, the label updating strategy, the label propagation strategy, and the convergence conditions. Compared with the primitive LPA, existing drawbacks are solved by CK-LPA. Experiments and benchmarks reveal that our proposed method sustains nearly linear time complexity and exhibits significant improvements in the quality aspect of static community detection. Hence, the algorithm can be applied in large-scale social networks.

Lin, Zhen; Zheng, Xiaolin; Xin, Nan; Chen, Deren



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

PubMed Central

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [13C6]glucose incubation to evaluate the native glycated proteins labeled with [12C6]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration. PMID:19955080

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



An optimized isotopic labelling strategy of isoleucine-?2 methyl groups for solution NMR studies of high molecular weight proteins.  


An efficient synthetic route is proposed to produce 2-hydroxy-2-ethyl-3-oxobutanoate for the specific labelling of Ile methyl-?(2) groups in proteins. The (2)H, (13)C-pattern of the biosynthetic precursor has been designed to optimize magnetization transfer, in large proteins, between these important structural probes and their corresponding backbone nuclei. PMID:21792424

Ayala, Isabel; Hamelin, Olivier; Amero, Carlos; Pessey, Ombeline; Plevin, Michael J; Gans, Pierre; Boisbouvier, Jérôme



Efficient mixing of the solar nebula from uniform Mo isotopic composition of meteorites.  


The abundances of elements and their isotopes in our Galaxy show wide variations, reflecting different nucleosynthetic processes in stars and the effects of Galactic evolution. These variations contrast with the uniformity of stable isotope abundances for many elements in the Solar System, which implies that processes efficiently homogenized dust and gas from different stellar sources within the young solar nebula. However, isotopic heterogeneity has been recognized on the subcentimetre scale in primitive meteorites, indicating that these preserve a compositional memory of their stellar sources. Small differences in the abundance of stable molybdenum isotopes in bulk rocks of some primitive and differentiated meteorites, relative to terrestrial Mo, suggest large-scale Mo isotopic heterogeneity between some inner Solar System bodies, which implies physical conditions that did not permit efficient mixing of gas and dust. Here we report Mo isotopic data for bulk samples of primitive and differentiated meteorites that show no resolvable deviations from terrestrial Mo. This suggests efficient mixing of gas and dust in the solar nebula at least to 3 au from the Sun, possibly induced by magnetohydrodynamic instabilities. These mixing processes must have occurred before isotopic fractionation of gas-phase elements and volatility-controlled chemical fractionations were established. PMID:12968172

Becker, Harry; Walker, Richard J




EPA Science Inventory

Oxygen-18 (18-O) labeling provides a sensitive means for quantifying oxygen binding that occurs during in vivo oxidations. Oxidants (ozone, nitrogen oxides, hydrogen peroxide, etc.) are first synthesized using 18-O, then cells or tissues are exposed to the labeled ...


Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins  

SciTech Connect

A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on /sup 15/N (or /sup 13/C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-/sup 15/N (or /sup 13/C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from /sup 15/N-bonded protons in the backbone and sidechains of uniformly /sup 15/N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-/sup 15/N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of /sup 15/N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple /sup 15/N and /sup 13/C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl(/sup 15/N)alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

McIntosh, L.P.; Griffey, R.H.; Muchmore, D.C.; Nielson, C.P.; Redfield, A.G.; Dahlquist, F.W.



Coral mucus stable isotope composition and labeling: experimental evidence for mucus uptake by epizoic acoelomorph worms  

Microsoft Academic Search

Mucus released by scleractinian corals can act as an important energy and nutrient carrier in coral reef ecosystems, and a\\u000a distinct isotopic signature would allow following the fate of this material. This study investigates the natural C and N stable\\u000a isotopic signatures of mucus released by four scleractinian coral genera (Acropora, Fungia, Pocillopora and Stylophora) in comparison with those of

Malik S. Naumann; Christoph Mayr; Ulrich Struck; Christian Wild



A software toolkit and interface for performing stable isotope labeling and top3 quantification using Progenesis LC-MS.  


Numerous software packages exist to provide support for quantifying peptides and proteins from mass spectrometry (MS) data. However, many support only a subset of experimental methods or instrument types, meaning that laboratories often have to use multiple software packages. The Progenesis LC-MS software package from Nonlinear Dynamics is a software solution for label-free quantitation. However, many laboratories using Progenesis also wish to employ stable isotope-based methods that are not natively supported in Progenesis. We have developed a Java programming interface that can use the output files produced by Progenesis, allowing the basic MS features quantified across replicates to be used in a range of different experimental methods. We have developed post-processing software (the Progenesis Post-Processor) to embed Progenesis in the analysis of stable isotope labeling data and top3 pseudo-absolute quantitation. We have also created export ability to the new data standard, mzQuantML, produced by the Proteomics Standards Initiative to facilitate the development and standardization process. The software is provided to users with a simple graphical user interface for accessing the different features. The underlying programming interface may also be used by Java developers to develop other routines for analyzing data produced by Progenesis. PMID:22888986

Qi, Da; Brownridge, Philip; Xia, Dong; Mackay, Katherine; Gonzalez-Galarza, Faviel F; Kenyani, Jenna; Harman, Victoria; Beynon, Robert J; Jones, Andrew R



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


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

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



Identification of proteolytic products and natural protein N-termini by Terminal Amine Isotopic Labeling of Substrates (TAILS).  


Determining the sequence of protein N-termini and their modifications functionally annotates proteins since translation isoforms, posttranslational modifications, and proteolytic truncations direct localization, activity, and the half-life of most proteins. Here we present in detail the steps required to perform our recently described approach we call Terminal Amine Isotopic Labeling of Substrates (TAILS), a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative and global analysis of the N-terminome and proteolysis in one MS/MS experiment. By a 3-day procedure with flexible ?- and ?-amine labeling and blocking options, TAILS removes internal tryptic and C-terminal peptides by binding to a dendritic polyglycerol aldehyde polymer. Therefore, by negative selection, this enriches for both the N-terminal-labeled peptides and all forms of naturally blocked N-terminal peptides. In addition to providing valuable proteome annotation, the simultaneous analysis of the original mature N-terminal peptides enables these peptides to be used for higher confidence protein substrate identification by two or more different and unique peptides. Second, the analysis of the N-terminal peptides forms a statistical classifier to determine valid isotope ratio cutoffs in order to identify with high-confidence protease-generated neo-N-terminal peptides. Third, quantifying the loss of acetylated or cyclized N-terminal peptides that have been cleaved extends overall substrate coverage. Hence, TAILS allows for the global analysis of the N-terminome and determination of cleavage site motifs and substrates for protease including those with unknown or broad specificity. PMID:21604129

Doucet, Alain; Kleifeld, Oded; Kizhakkedathu, Jayachandran N; Overall, Christopher M



Use of a new gas chromatograph isotope ratio mass spectrometer to trace exogenous 13 C labelled glucose at a very low level of enrichment in man  

Microsoft Academic Search

Summary  The use of 13C labelled glucose in human metabolic studies has been limited by the high cost of the tracer and the problems of measuring low 13C isotopic abundance in plasma glucose. In the present work we describe a new gas chromatograph-isotope ratio mass spectrometer allowing the measurement of a 0.001 atom % increase in 13C abundance over baseline, on

S. Tissot; S. Normand; R. Guilluy; C. Pachiaudi; M. Beylot; M. Laville; R. Cohen; R. Mornex; J. P. Riou



Sulfur-34S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34) of Proteomic Changes in Pseudomonas fluorescens during Naphthalene Degradation*  

PubMed Central

The relative quantification of proteins is one of the major techniques used to elucidate physiological reactions. Because it allows one to avoid artifacts due to chemical labeling, the metabolic introduction of heavy isotopes into proteins and peptides is the preferred method for relative quantification. For eukaryotic cells, stable isotope labeling by amino acids in cell culture (SILAC) has become the gold standard and can be readily applied in a vast number of scenarios. In the microbial realm, with its highly versatile metabolic capabilities, SILAC is often not feasible, and the use of other 13C or 15N labeled substrates might not be practical. Here, the incorporation of heavy sulfur isotopes is shown to be a useful alternative. We introduce 34S stable isotope labeling of amino acids for quantification and the corresponding tools required for spectra extraction and disintegration of the isotopic overlaps caused by the small mass shift. As proof of principle, we investigated the proteomic changes related to naphthalene degradation in P. fluorescens ATCC 17483 and uncovered a specific oxidative-stress-like response. PMID:23603340

Herbst, Florian-Alexander; Taubert, Martin; Jehmlich, Nico; Behr, Tobias; Schmidt, Frank; von Bergen, Martin; Seifert, Jana



Automated LC-HRMS(/MS) approach for the annotation of fragment ions derived from stable isotope labeling-assisted untargeted metabolomics.  


Structure elucidation of biological compounds is still a major bottleneck of untargeted LC-HRMS approaches in metabolomics research. The aim of the present study was to combine stable isotope labeling and tandem mass spectrometry for the automated interpretation of the elemental composition of fragment ions and thereby facilitate the structural characterization of metabolites. The software tool FragExtract was developed and evaluated with LC-HRMS/MS spectra of both native (12)C- and uniformly (13)C (U-(13)C)-labeled analytical standards of 10 fungal substances in pure solvent and spiked into fungal culture filtrate of Fusarium graminearum respectively. Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment. The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-(13)C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion. Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies. Moreover, as demonstrated for a fungal culture sample, FragExtract also assists the characterization of unknown metabolites, which are not contained in databases, and thus exhibits a significant contribution to untargeted metabolomics research. PMID:24965664

Neumann, Nora K N; Lehner, Sylvia M; Kluger, Bernhard; Bueschl, Christoph; Sedelmaier, Karoline; Lemmens, Marc; Krska, Rudolf; Schuhmacher, Rainer



Regional cooperation in energy efficiency standard-setting and labeling in North America  

SciTech Connect

The North American Energy Working Group (NAEWG) was established in 2001 by the governments of Canada, Mexico, and the United States. The goals of NAEWG are to foster communication and cooperation on energy-related matters of common interest, and to enhance North American energy trade and interconnections consistent with the goal of sustainable development, for the benefit of all three countries. At its outset, NAEWG established teams to address different aspects of the energy sector. One, the Energy Efficiency Expert Group, undertook activity in three areas: (1) analyzing commonalities and differences in the test procedures of Canada, Mexico, and the United States, and identifying specific products for which the three countries might consider harmonization; (2) exploring possibilities for increased mutual recognition of laboratory test results; and (3) looking at possibilities for enhanced cooperation in the Energy Star voluntary endorsement labeling program. To support NAEWG's Expert Group on Energy Efficiency (NAEWG-EE), USDOE commissioned Lawrence Berkeley National Laboratory, representing the Collaborative Labeling and Appliance Standards Program (CLASP), to prepare a resource document comparing current standards, labels, and test procedure regulations in Canada, Mexico, and the United States. The resulting document identified 46 energy-using products for which at least one of the three countries has energy efficiency regulations. Three products--refrigerators/freezers, room air conditioners, and integral horsepower three-phase electric motors--have identical minimum energy performance standards (MEPS) and test procedures in the three countries. Ten other products have different MEPS and test procedures, but have the near-term potential for harmonization. NAEWG-EE is currently working to identify mechanisms for mutual recognition of test results. With consultative support from the United States and Canada through NAEWG-EE, Mexico is exploring possibilities for extending the Energy Star endorsement label to Mexico.

Wiel, Stephen; Van Wie McGrory, Laura



Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria  

PubMed Central

Summary Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [2H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC–MS. Feeding experiments with [2H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [2H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [2H3]methionine and 34SO4 2?, synthesized from elemental 34S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction. PMID:23766810

Brock, Nelson L; Citron, Christian A; Zell, Claudia; Berger, Martine; Wagner-Dobler, Irene; Petersen, Jorn; Brinkhoff, Thorsten; Simon, Meinhard



Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization  

PubMed Central

An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S?-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency. PMID:21952753

Lu, Yali; Zhou, Xiao; Stemmer, Paul M.; Reid, Gavin E.



Efficient labelling of enzymatically synthesized vinyl-modified DNA by an inverse-electron-demand Diels-Alder reaction.  


Many applications in biotechnology and molecular biology rely on modified nucleotides. Here, we present an approach for the postsynthetic labelling of enzymatically synthesized vinyl-modified DNA by Diels-Alder reaction with inverse electron demand using a tetrazine. Labelling proceeds very efficiently and supersedes several known approaches. PMID:25089682

Busskamp, Holger; Batroff, Ellen; Niederwieser, Andrea; Abdel-Rahman, Obadah S; Winter, Rainer F; Wittmann, Valentin; Marx, Andreas



Stable Isotopic Labeling by Amino Acids in Cultured Primary Neurons: Application to Brain-derived Neurotrophic Factor-dependent Phosphotyrosine-associated Signaling  

Microsoft Academic Search

Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we demon- strated that stable isotope labeling by amino acids in cell culture (SILAC) can be applied to a differentiated, non- dividing cell type such as primary neurons, and we applied this technique to assess changes in the neuronal phos- photyrosine proteome in

Daniel S. Spellman; Katrin Deinhardt; Costel C. Darie; Moses V. Chao; Thomas A. Neubert



Stable Isotope Metabolic Labeling with a Novel 15N-Enriched Bacteria Diet for Improved Proteomic Analyses of Mouse Models for Psychopathologies  

Microsoft Academic Search

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the

Elisabeth Frank; Melanie S. Kessler; Michaela D. Filiou; Yaoyang Zhang; Giuseppina Maccarrone; Stefan Reckow; Mirjam Bunck; Hermann Heumann; Christoph W. Turck; Rainer Landgraf; Boris Hambsch



Sedimented cyanobacterial detritus as a source of nutrient for submerged macrophytes (Vallisneria spiralis and Elodea nuttallii): An isotope labeling experiment using 15N  

Microsoft Academic Search

A tracer experiment using the nitrogen isotope 15N investigated the uptake and incorporation of nitrogen from sedimented cyanobacterial detritus by two species of submerged macrophytes, the native Vallisneria spiralis and the exotic Elodea nuttallii, in Lake Taihu (China). The cyanobacterium Microcystis was labeled with 15Nammonium and dried to produce detritus, which was injected into vegetated sediments and traced to establish

Leiyan Zhang; Kuanyi Li; Zhengwen Liu; Jack J. Middelburg



Thermal desorption studies of isotopically-labeled oxygen-induced superconductivity in La2CuO(4+delta)  

NASA Astrophysics Data System (ADS)

Isotopically-labeled oxygen enrichment and thermal desorption mass spectroscopy (TDMS) have been combined to study interstitial oxygen desorption from superconducting La2CuO(4+delta) (delta less than or equal to 0.032). Single crystal samples of magnetic insulating La2CuO(4.00) were annealed at 860 K under 1-3 kbar oxygen pressure for 12-100 hours to yield hole-doped, superconducting La2CuO(4+delta) samples with Tc between 35 and 40 K. Whereas no TDMS signals were observed for the insulator, rapid bursts (full width at half maximum less than 0.5 sec) of molecular oxygen were observed above 350 K while heating the superconductor at less than 1 K per sec in high vacuum. A kinetic model is proposed in which the interstitial oxygen diffuses to internal grain boundaries and defects during heating, thereby inducing stress in the lattice as it attempts to revert to the LaCuO(4.00) crystal structure. This stress is relieved by lattice fracture at grain boundaries during the TDMS experiment, releasing the trapped oxygen from the sample as micro-cracks are formed. In addition, the facile oxygen exchange between interstitial and lattice oxygen sites has been discovered by TDMS and weight gain measurements from isotopically-enriched crystals, supporting the structural model of Chaillout, et al. in which the interstitial oxygen atom dimerizes with a lattice oxygen ion.

Shinn, N. D.; Bartram, M. E.; Schirber, J. E.; Overmyer, D. L.; Rogers, J. W., Jr.; Fisk, Z.; Cheong, S. W.


Dynamic Redistribution of Isotopically Labeled Cohorts of Nitrogen Inputs in Two Temperate Forests  

Microsoft Academic Search

We compared simulated time series of nitrogen-15 (15N) redistribution following a large-scale labeling experiment against field recoveries of 15NH41 and 15NO32 in vegetation tissues. We sought to gain insight into the altered modes of N cycling under long-term, experimentally elevated N inputs. The study took place in two contrasting forests: a red pine stand and a mixed deciduous stand (predomi-

William S. Currie; Knute J. Nadelhoffer


Original Articles: Dynamic Redistribution of Isotopically Labeled Cohorts of Nitrogen Inputs in Two Temperate Forests  

Microsoft Academic Search

We compared simulated time series of nitrogen-15 (15N) redistribution following a large-scale labeling experiment against field recoveries of 15NH4\\u000a + and\\u000a 15NO3\\u000a ? in vegetation tissues. We sought to gain insight into the altered modes of N cycling under long-term, experimentally elevated\\u000a N inputs. The study took place in two contrasting forests: a red pine stand and a mixed deciduous

William S. Currie; Knute J. Nadelhoffer



International Review of the Development and Implementation of Energy Efficiency Standards and Labeling Programs  

SciTech Connect

Appliance energy efficiency standards and labeling (S&L) programs have been important policy tools for regulating the efficiency of energy-using products for over 40 years and continue to expand in terms of geographic and product coverage. The most common S&L programs include mandatory minimum energy performance standards (MEPS) that seek to push the market for efficient products, and energy information and endorsement labels that seek to pull the market. This study seeks to review and compare some of the earliest and most well-developed S&L programs in three countries and one region: the U.S. MEPS and ENERGY STAR, Australia MEPS and Energy Label, European Union MEPS and Ecodesign requirements and Energy Label and Japanese Top Runner programs. For each program, key elements of S&L programs are evaluated and comparative analyses across the programs undertaken to identify best practice examples of individual elements as well as cross-cutting factors for success and lessons learned in international S&L program development and implementation. The international review and comparative analysis identified several overarching themes and highlighted some common factors behind successful program elements. First, standard-setting and programmatic implementation can benefit significantly from a legal framework that stipulates a specific timeline or schedule for standard-setting and revision, product coverage and legal sanctions for non-compliance. Second, the different MEPS programs revealed similarities in targeting efficiency gains that are technically feasible and economically justified as the principle for choosing a standard level, in many cases at a level that no product on the current market could reach. Third, detailed survey data such as the U.S. Residential Energy Consumption Survey (RECS) and rigorous analyses provide a strong foundation for standard-setting while incorporating the participation of different groups of stakeholders further strengthen the process. Fourth, sufficient program resources for program implementation and evaluation are critical to the effectiveness of standards and labeling programs and cost-sharing between national and local governments can help ensure adequate resources and uniform implementation. Lastly, check-testing and punitive measures are important forms of enforcement while the cancellation of registration or product sales-based fines have also proven effective in reducing non-compliance. The international comparative analysis also revealed the differing degree to which the level of government decentralization has influenced S&L programs and while no single country has best practices in all elements of standards and labeling development and implementation, national examples of best practices for specific elements do exist. For example, the U.S. has exemplified the use of rigorous analyses for standard-setting and robust data source with the RECS database while Japan?s Top Runner standard-setting principle has motivated manufacturers to exceed targets. In terms of standards implementation and enforcement, Australia has demonstrated success with enforcement given its long history of check-testing and enforcement initiatives while mandatory information-sharing between EU jurisdictions on compliance results is another important enforcement mechanism. These examples show that it is important to evaluate not only the drivers of different paths of standards and labeling development, but also the country-specific context for best practice examples in order to understand how and why certain elements of specific S&L programs have been effective.

Zhou, Nan; Zheng, Nina; Fridley, David



Field-scale isotopic labeling of phospholipid fatty acids from acetate-degrading sulfate-reducing bacteria.  


Isotopic labeling of biomarker molecules is a technique applied to link microbial community structure with activity. Previously, we successfully labeled phospholipid fatty acids (PLFA) of suspended nitrate-reducing bacteria in an aquifer. However, the application of the method to low energy-yielding processes such as sulfate reduction, and extension of the analysis to attached communities remained to be studied. To test the feasibility of the latter application, an anoxic test solution of 500 l of groundwater with addition of 0.5 mM Br- as a conservative tracer, 1.1 mM SO4(2-), and 2.0 mM [2-13C]acetate was injected in the transition zone of a petroleum hydrocarbon-contaminated aquifer where sulfate-reducing and methanogenic conditions prevailed. Thousand liters of test solution/groundwater mixture were extracted in a stepwise fashion after 2-46 h incubation. Computed apparent first-order rate coefficients were 0.31+/-0.04 day(-1) for acetate and 0.34+/-0.05 day(-1) for SO4(2-) consumption. The delta13C increased from -71.03 per thousand to +3352.50 per thousand in CH4 and from -16.15 per thousand to +32.13 per thousand in dissolved inorganic carbon (DIC). A mass balance suggested that 43% of the acetate-derived (13)C appeared in DIC and 57% appeared in CH4. Thus, acetate oxidation coupled to sulfate reduction and acetoclastic methanogenesis occurred simultaneously. The delta13C of PLFA increased on average by 27 per thousand in groundwater samples and 4 per thousand in sediment samples. Hence, both suspended and attached communities actively degraded acetate. The PLFA labeling patterns and fluorescent in situ hybridization (FISH) analyses of sediment and groundwater samples suggested that the main sulfate-reducing bacteria degrading the acetate were Desulfotomaculum acetoxidans and Desulfobacter sp. in groundwater, and D. acetoxidans in sediment. PMID:16329868

Pombo, Silvina A; Kleikemper, Jutta; Schroth, Martin H; Zeyer, Josef



Human lactation: maternal transfer of dietary triglycerides labeled with stable isotopes  

SciTech Connect

A stable isotope tracer method was utilized to measure quantitatively the secretion of diet-derived fatty acids (FA) into human milk. A mixture of (/sup 2/H6)tripalmitin, (/sup 2/H18)-triolein, and (/sup 2/H12)trilinolein was administered to three healthy, lactating women 22 to 30 years of age. Milk and blood samples were collected sequentially for 72 hr. The FA composition and concentration of total plasma, lipoprotein, and milk triglycerides were determined by gas-liquid chromatography (GLC) and the isotopic enrichment was determined by gas-liquid chromatography-mass spectrometry (GLC-MS). There were no statistically significant differences in mammary secretion of the individual fats, either by a single individual or between subjects. The mean secretion of fat by one breast was 5.11 +/- 1.26% of the dose (CV = 25%). There was a significant 6.0-hr delay between peak occurrence of the tracer in plasma and its occurrence in milk. The lipids are transported to the mammary gland primarily by the chylomicron and very low density lipoprotein triglycerides.

Hachey, D.L.; Thomas, M.R.; Emken, E.A.; Garza, C.; Brown-Booth, L.; Adlof, R.O.; Klein, P.D.



Competition for phosphorus: differential uptake from dual-isotope--labeled soil interspaces between shrub and grass.  


Two species of Agropyron grass differed strikingly in their capacity to compete for phosphate in soil interspaces shared with a common competitor, the sagebrush Artemisia tridentata. Of the total phosphorus-32 and -33 absorbed by Artemisia, 86 percent was from the interspace shared with Agropyron spicatum and only 14 percent from that shared with Agropyron desertorum. Actively absorbing mycorrhizal roots of Agropyron and Artemisia were present in both interspaces, where competition for the labeled phosphate occurred. The results have important implications about the way in which plants compete for resources below ground in both natural plant communities and agricultural intercropping systems. PMID:17795898

Caldwell, M M; Eissenstat, D M; Richards, J H; Allen, M F



Competition for phosphorus: differential uptake from dual-isotope-labeled soil interspaces between shrub and grass  

SciTech Connect

Two species of Agropyron grass differed strikingly in their capacity to compete for phosphate in soil interspaces shared with a common competitor, the sagebrush Artemisia tridentata. Of the total phosphorus-32 and -33 absorbed by Artemisia, 86% was from the interspace shared with Agropyron spicatum and only 14% from that shared with Agropyron desertorum. Actively absorbing mycorrhizal roots of Agropyron and Artemisia were present in both interspaces, where competition for the labeled phosphate occurred. The results have important implications about the way in which plants compete for resources below ground in both natural plant communities and agricultural intercropping systems.

Caldwell, M.M.; Eissenstat, D.M.; Richards, J.H.; Allen, M.F.



An Efficient Method to Calculate the Aggregated Isotopic Distribution and Exact Center-Masses  

NASA Astrophysics Data System (ADS)

In this article, we present a computation- and memory-efficient method to calculate the probabilities of occurrence and exact center-masses of the aggregated isotopic distribution of a molecule. The method uses fundamental mathematical properties of polynomials given by the Newton-Girard theorem and Viete's formulae. The calculation is based on the atomic composition of the molecule and the natural abundances of the elemental isotopes in normal terrestrial matter. To evaluate the performance of the proposed method, which we named BRAIN, we compare it with the results obtained from five existing software packages ( IsoPro, Mercury, Emass, NeutronCluster, and IsoDalton) for 10 biomolecules. Additionally, we compare the computed mass centers with the results obtained by calculating, and subsequently aggregating, the fine isotopic distribution for two of the exemplary biomolecules. The algorithm will be made available as a Bioconductor package in R, and is also available upon request.

Claesen, Jürgen; Dittwald, Piotr; Burzykowski, Tomasz; Valkenborg, Dirk



Combining capillary electrophoresis matrix-assisted laser desorption/ionization mass spectrometry and stable isotopic labeling techniques for comparative crustacean peptidomics.  


Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI-MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides. PMID:20334868

Wang, Junhua; Zhang, Yuzhuo; Xiang, Feng; Zhang, Zichuan; Li, Lingjun



Combining Capillary Electrophoresis Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Stable Isotopic Labeling Techniques for Comparative Crustacean Peptidomics  

PubMed Central

Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides. PMID:20334868

Wang, Junhua; Zhang, Yuzhuo; Xiang, Feng; Zhang, Zichuan; Li, Lingjun



iMS2Flux – a high–throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis  

PubMed Central

Background Metabolic flux analysis has become an established method in systems biology and functional genomics. The most common approach for determining intracellular metabolic fluxes is to utilize mass spectrometry in combination with stable isotope labeling experiments. However, before the mass spectrometric data can be used it has to be corrected for biases caused by naturally occurring stable isotopes, by the analytical technique(s) employed, or by the biological sample itself. Finally the MS data and the labeling information it contains have to be assembled into a data format usable by flux analysis software (of which several dedicated packages exist). Currently the processing of mass spectrometric data is time-consuming and error-prone requiring peak by peak cut-and-paste analysis and manual curation. In order to facilitate high-throughput metabolic flux analysis, the automation of multiple steps in the analytical workflow is necessary. Results Here we describe iMS2Flux, software developed to automate, standardize and connect the data flow between mass spectrometric measurements and flux analysis programs. This tool streamlines the transfer of data from extraction via correction tools to 13C-Flux software by processing MS data from stable isotope labeling experiments. It allows the correction of large and heterogeneous MS datasets for the presence of naturally occurring stable isotopes, initial biomass and several mass spectrometry effects. Before and after data correction, several checks can be performed to ensure accurate data. The corrected data may be returned in a variety of formats including those used by metabolic flux analysis software such as 13CFLUX, OpenFLUX and 13CFLUX2. Conclusion iMS2Flux is a versatile, easy to use tool for the automated processing of mass spectrometric data containing isotope labeling information. It represents the core framework for a standardized workflow and data processing. Due to its flexibility it facilitates the inclusion of different experimental datasets and thus can contribute to the expansion of flux analysis applications. PMID:23146204



In Vivo Stable Isotope Labeling of Fruit Flies Reveals Post-transcriptional Regulation in the Maternal-to-zygotic Transition*  

PubMed Central

An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that ?500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development. PMID:19321433

Gouw, Joost W.; Pinkse, Martijn W. H.; Vos, Harmjan R.; Moshkin, Yuri; Verrijzer, C. Peter; Heck, Albert J. R.; Krijgsveld, Jeroen



An efficient simulator for pinhole imaging of PET isotopes.  


Today, small-animal multi-pinhole single photon emission computed tomography (SPECT) can reach sub-half-millimeter image resolution. Recently we have shown that dedicated multi-pinhole collimators can also image PET tracers at sub-mm level. Simulations play a vital role in the design and optimization of such collimators. Here we propose and validate an efficient simulator that models the whole imaging chain from emitted positron to detector signal. This analytical simulator for pinhole positron emission computed tomography (ASPECT) combines analytical models for pinhole and detector response with Monte Carlo (MC)-generated kernels for positron range. Accuracy of ASPECT was validated by means of a MC simulator (MCS) that uses a kernel-based step for detector response with an angle-dependent detector kernel based on experiments. Digital phantom simulations with ASPECT and MCS converge to almost identical images. However, ASPECT converges to an equal image noise level three to four orders of magnitude faster than MCS. We conclude that ASPECT could serve as a practical tool in collimator design and iterative image reconstruction for novel multi-pinhole PET. PMID:21335647

Goorden, M C; van der Have, F; Kreuger, R; Beekman, F J



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

SciTech Connect

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

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



Advanced Identification of Proteins in Uncharacterized Proteomes by Pulsed in Vivo Stable Isotope Labeling-based Mass Spectrometry*  

PubMed Central

Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized. This problem is aggravated by the presence of large numbers of expressed sequence tag clones that lack homologues in other species, which makes it difficult to identify new proteins irrespective of whether such molecules are involved in species-specific biological processes. We have used a pulsed stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry method, which is based on the detection of paired peptides after [13C6]lysine incorporation into proteins in vivo, to greatly increase the confidence of protein identification in cross-species database searches. The method was applied to identify nearly 3000 proteins in regenerating tails of the urodele amphibian Notophthalmus viridescens, which possesses outstanding capabilities in the regeneration of complex tissues. We reason that pulsed in vivo SILAC represents a versatile tool to identify new proteins in species for which only limited sequence information exists. PMID:20139370

Looso, Mario; Borchardt, Thilo; Kruger, Marcus; Braun, Thomas



Stable isotope dimethyl labeling combined with LTQ mass spectrometric detection, a quantitative proteomics technology used in liver cancer research  

PubMed Central

Liver cancer is a common malignant disease, with high incidence and mortality rates. The study on the proteomics of liver cancer has attracted particular attention. The quantitative study method of proteomics depends predominantly on two-dimensional (2D) gel electrophoresis. In the present study we reported a rapid and accurate proteomics quantitative study method of high repeatability that includes the use of stable isotope labeling for the extraction of proteins and peptides via enzymolysis to achieve new type 2D capillary liquid chromatography-mass spectrometry separation using the separation mode of cation-exchange chromatography in conjunction with reversed-phase chromatography. LTQ OrbiTrap mass spectrometry detection was also performed. A total of 188 differential proteins were analyzed, including 122 upregulating [deuterium/hydrogen ratio (D/H) >1.5)] and 66 downregulating proteins (D/H<0.67). These proteins may play an important role in the occurrence, drug resistance, metastasis and recurrence of cancer or other pathological processes. Such a proteomics technology may provide biological data as well as a new methodological basis for liver cancer research. PMID:24648984




Efficient laser isotope separation of 13C using novel linear multi-pass cavity  

NASA Astrophysics Data System (ADS)

13C isotope has been separated in the form of enriched product C2F4 by selective multi-photon dissociation (MPD) of Freon-22 (CHClF2) using the 9P(26) laser line of a transversely excited atmospheric CO2 laser. The non-linearity factor, ?, that determines the dependence of the yield of 13C isotope on the fluence (J/cm2) has been determined for various laser rotational lines (9P(20) 9P(26)) and the advantage of a lower ? in the case of 9P(26) is highlighted for macroscopic production of 13C isotope. It is also shown that a higher value of the optimum fluence at 9P(26) not only results in a higher enrichment efficiency but in a relatively lower value of ? also. The laser pulse energy is efficiently utilized for selective MPD of Freon-22 by focusing the pulse energy repeatedly with the help of a novel linear multi-pass cavity (LMPC). The novelty of this optical arrangement lies in its ability to maintain the laser fluence around an optimum value for a desired enrichment of 13C in the product. This also ensures a higher quantity of enriched product because of the higher reaction volume. The advantage of the LMPC over the conventionally used Herriott multi-pass cell has also been presented. The gain in reaction volume in the present optical cavity having 20 passes with a constant fluence in each pass is as high as 12. Isotope-selective MPD of Freon in a LMPC with constant fluence in each pass showed a distinct advantage in energy utilization to separate 13C isotope over the gradually reducing fluence case.

Kumar, M.; Gupta, V.; Nath, A. K.



Overcoming matrix effects in electrospray: quantitation of ?-agonists in complex matrices by isotope dilution liquid chromatography-mass spectrometry using singly (13)C-labeled analogues.  


In this work, the implementation of isotope dilution mass spectrometry (IDMS) using minimal labeling and isotope pattern deconvolution (IPD) is evaluated as a strategy for the minimization of matrix effects during trace determination of ?2-agonists in complex matrices by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). First, the parameters affecting the measurement of isotopic composition of organic compounds by liquid chromatography electrospray ionization high resolution mass spectrometry with a time-of-flight analyzer were evaluated using as a case of study three different ?2-agonists: clenbuterol, clenproperol and brombuterol. Then, a calibration graph-free IDMS methodology was evaluated in order to overcome matrix effects in LC-ESI-MS in complex samples. In this procedure singly (13)C-labeled analogues of clenbuterol, clenproperol and brombuterol were employed in combination with IPD. Using this approach accurate and precise results were obtained in the simultaneous quantification of ?2-agonists in human urine and bovine liver, even at the sub ngg(-1) and particularly in spite of the previously reported matrix effects. Recovery rates in the range of 97-114% in fortified human urine and from 95% to 111% in fortified bovine liver were obtained with RSD (%) of independent recovery experiments always lower than 6%. These results demonstrate that the proposed methodology based on the use of (13)C1-labeled standards and IPD is a reliable approach for accurate LC-MS quantitation of small molecules and compatible with full-scan high-resolution mass spectrometry. PMID:23523066

González-Antuña, Ana; Domínguez-Romero, Juan C; García-Reyes, Juan F; Rodríguez-González, Pablo; Centineo, Giuseppe; García Alonso, J Ignacio; Molina-Díaz, Antonio



Effect of microwave irradiation on antibody labeling efficiency when applied to ultrathin cryosections through fixed biological material.  


To study the effect of microwaves on immunolabeling, ultrathin cryosections or diluted antibodies were irradiated prior to antibody application, and gold labeling was quantified. In addition, affinity purified, polyclonal antibodies and protein A-gold were applied to ultrathin cryosections of aldehyde-fixed material in the presence and absence of microwaves. Amylase, a soluble protein secreted by pancreatic acinar cells, MHC class II, an integral membrane protein, and 3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine (DAMP), an exogenously added antigen, were localized with monospecific antibodies. Each was chosen for their contrasting subcellular characteristics. Results demonstrated that for some antigens, antibody labeling efficiency was quantitatively improved by microwave irradiation of sections prior to antibody application. Irradiation of diluted antibodies prior to their application also resulted in improved labeling. In contrast, the results obtained using rapid immunolabeling protocols in the presence of microwaves resulted in labeling levels similar to those obtained in the absence of microwaves. We conclude that microwave irradiation can improve the labeling efficiency of some antibodies. However, improvements in labeling density are dependent on the antigen under study and on variable irradiation times, unique to each antibody. This suggests that the routine use of microwaves to reduce incubation times may not be a viable alternative to bench protocols. PMID:9712160

Chicoine, L; Webster, P



An optimal defense strategy for phenolic glycoside production in Populus trichocarpa--isotope labeling demonstrates secondary metabolite production in growing leaves.  


Large amounts of carbon are required for plant growth, but young, growing tissues often also have high concentrations of defensive secondary metabolites. Plants' capacity to allocate resources to growth and defense is addressed by the growth-differentiation balance hypothesis and the optimal defense hypothesis, which make contrasting predictions. Isotope labeling can demonstrate whether defense compounds are synthesized from stored or newly fixed carbon, allowing a detailed examination of these hypotheses. Populus trichocarpa saplings were pulse-labeled with 13CO2 at the beginning and end of a growing season, and the 13C signatures of phenolic glycosides (salicinoids), sugars, bulk tissue, and respired CO2 were traced over time. Half of the saplings were also subjected to mechanical damage. Populus trichocarpa followed an optimal defense strategy, investing 13C in salicinoids in expanding leaves directly after labeling. Salicinoids turned over quickly, and their production continued throughout the season. Salicin was induced by early-season damage, further demonstrating optimal defense. Salicinoids appear to be of great value to P. trichocarpa, as they command new C both early and late in the growing season, but their fitness benefits require further study. Export of salicinoids between tissues and biochemical pathways enabling induction also needs research. Nonetheless, the investigation of defense production afforded by isotope labeling lends new insights into plants' ability to grow and defend simultaneously. PMID:24739022

Massad, Tara Joy; Trumbore, Susan E; Ganbat, Gantsetseg; Reichelt, Michael; Unsicker, Sybille; Boeckler, Andreas; Gleixner, Gerd; Gershenzon, Jonathan; Ruehlow, Steffen



Convex onion peeling genetic algorithm: an efficient solution to map labeling of point-feature  

Microsoft Academic Search

Map labeling of point-feature is the problem of placing text labels to corresponding point features on a map in a way that minimizes overlaps while satisfying basic rules for the quality. This problem is a critical problem in the applica- tions of cartography and Geographical Information Systems (GIS). In this paper we study the fundamental issues related to map labeling

Wan D. Bae; Shayma Alkobaisi; Petr Vojtechovský; Sada Narayanappa; Kye Y. Bae



An Optimized Method for Computing 18O/16O Ratios of Differentially Stable-isotope Labeled Peptides in the Context of Post-digestion 18O Exchange/Labeling  

PubMed Central

Differential 18O/16O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H218O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs, has made trypsin-catalyzed 18O post-digestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed 18O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the 18O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual 18O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then 18O/16O ratios derived via evolutionary programming. The algorithm is tested using trypsin–catalyzed 18O post-digestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both, accuracy and precision are improved utilizing this rigorous mathematical approach. Utilizing this algorithmic technique, we demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios for differentially labeled BSA peptides, by accounting for artifacts caused by a variable degree of post-digestion 18O exchange. We further demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its non myristylated mutant. PMID:20540505

Ye, Xiaoying; Luke, Brian T.; Johann, Donald J.; Ono, Akira; Chan, King C.; Prieto, DaRue A.; Issaq, Haleem J.; Veenstra, Timothy D.; Blonder, Josip



Quantitative determination of free and total bisphenol A in human urine using labeled BPA glucuronide and isotope dilution mass spectrometry.  


Bisphenol A (BPA) is a widely used industrial chemical in the manufacturing of polycarbonate plastic bottles, food and beverage can linings, thermal receipts, and dental sealants. Animal and human studies suggest that BPA may disrupt normal hormonal function and hence, potentially, have negative effects on the human health. While total BPA is frequently reported, it is recognized that free BPA is the biologically active form and is rarely reported in the literature. The objective of this study was to develop a sensitive and improved method for the measurement of free and total BPA in human urine. Use of a labeled conjugated BPA (bisphenol A-d6 ?-D-glucuronide) allowed for the optimization of the enzymatic reaction and permitted an accurate determination of the conjugated BPA concentration in urine samples. In addition, a (13)C12-BPA internal standard was used to account for the analytical recoveries and performance of the isotope dilution method. Solid-phase extraction (SPE) combined with derivatization and analysis using a triple quadrupole GC-EI/MS/MS system achieved very low method detection limit of 0.027 ng/mL. BPA concentrations were measured in urine samples collected during the second and third trimesters of pregnancy in 36 Canadian women. Total maternal BPA concentrations in urine samples ranged from not detected to 9.40 ng/mL (median, 1.21 ng/mL), and free BPA concentrations ranged from not detected to 0.950 ng/mL (median, 0.185 ng/mL). Eighty-six percent of the women had detectable levels of conjugated BPA, whereas only 22 % had detectable levels of free BPA in their urine. BPA levels measured in this study agreed well with data reported internationally. PMID:24817354

Kubwabo, Cariton; Kosarac, Ivana; Lalonde, Kaela; Foster, Warren G



(34)S and (15)N labelling to model S and N flux in plants and determine the different components of N and S use efficiency.  


In order to highlight our understanding on ecosystems functioning and resource sharing/competition, either in artificial environment or agrosystems, according to changes in the climatic conditions, it is necessary to measure accurately element fluxes within plants. Stable isotopes allow tracking safely and accurately on a short time frame the behavior of elements in plants. After a short review devoted to isotopic studies of elemental flux within plants, we explain how a direct multiple labelling study might be conducted in a plant, so as to measure over short time nitrogen and sulfur acquisition, and assimilates arising from a labelled source. PMID:24222425

Salon, Christophe; Bataillé, Marie-Paule; Gallardo, Karine; Jeudy, Christian; Santoni, Anne-Lise; Trouverie, Jacques; Voisin, Anne-Sophie; Avice, Jean-Christophe



Transpiration efficiency over an annual cycle, leaf gas exchange and wood carbon isotope ratio of three tropical tree species  

E-print Network

Transpiration efficiency over an annual cycle, leaf gas exchange and wood carbon isotope ratio; published online August 6, 2009 Summary Variation in transpiration efficiency (TE) and its relationship. Cumulative transpiration was determined by repeatedly weighing the pots with a pallet truck scale. Dry matter

Bermingham, Eldredge


A Simple and Efficient Algorithm for High-Quality Line Labeling  

E-print Network

. In order to formalize what good line labeling means, we studied Imhof's rules for positioning names on maps (Imhof, 1975). His well-established catalogue of label placement rules also provides a set of guidelines (van Dijk et al., 1999).) Imhof's rules can be put into two categories, namely hard and soft

Utrecht, Universiteit



Microsoft Academic Search

This paper put forward an new solution, which adopted the genetic algorithm to obtain the global optimal solution (approximate) of automated label placement of point feature. In the paper, the basic thought and design framework of using genetic algorithm to solve point feature labelling was firstly introduced, then, some practical technique and new improved method during the experiment procedure of

Fan Hong; Liu Kaijun; Zhang Zuxun



Microsoft Academic Search

This paper put forward an new solution, which adopted the genetic algorithm to obtain the global optimal solution (approximate) of automated label placement of point feature. In the paper, the basic thought and design framework of using genetic algorithm to solve point feature labelling was firstly introduced, then, some practical technique and new improved method during the experiment procedure of

Fan Hong; Liu Kaijun; Zhang Zuxun


An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.  


Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. PMID:24662061

Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin



Quantitative amino acid profiling and stable isotopically labeled amino acid tracer enrichment used for in vivo human systemic and tissue kinetics measurements.  


An important area within clinical functional metabolomics is in vivo amino acid metabolism and protein turnover measurements for which accurate amino acid concentrations and stable isotopically labeled amino acid enrichments are mandatory not the least when tissue metabolomics is determined. The present study describes a new sensitive liquid chromatography tandem mass-spectrometry method quantifying 20 amino acids and their tracer(s) ([ring-(13)C6]/D5Phenylalanine) in human plasma and skeletal muscle specimens. Before analysis amino acids were extracted and purified via deprotonization/ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-(13)C/(15)N). The method was validated according to general recommendations for chromatographic analytical methods. The calibration curve correlations for amino acids were on average; r(2)=0.998. Interday accuracy for amino acids determined in spiked plasma was on average 97.3% and the coefficient of variation (CV) was 2.6%. The ([ring-(13)C6]/D5Phenylalanine) enrichment CV's for machine reproducibility in muscle tissue fluid and plasma were 4.4 and 0.8%, and the interday variability was 3.4% and the recovery was 90.5%, respectively. In conclusion, we have developed and validated a method for quantitative amino acid profiling that meets the requirements for systemic and tissue human in vivo amino acid and protein turnover kinetics measurements. Moreover, citrulline, ornithine, ?-methyl-histidine, ?-methyl-l-histidine, hydroxy-proline and carnitine were analysed but when similar precision and accuray are required an additional stable istopically labeled internal standard for these meatablites should be be added. PMID:24513911

Bornø, Andreas; van Hall, Gerrit



Prediction of equilibrium Li isotope fractionation between minerals and aqueous solutions at high P and T: An efficient ab initio approach  

Microsoft Academic Search

The mass-dependent equilibrium stable isotope fractionation between different materials is an important geochemical process. Here we present an efficient method to compute the isotope fractionation between complex minerals and fluids at high pressure, P, and temperature, T, representative for the Earth’s crust and mantle. The method is tested by computation of the equilibrium fractionation of lithium isotopes between aqueous fluids

Piotr M. Kowalski; Sandro Jahn



Primary Productivity Rates at Station ALOHA Determined by 18O Labeling and the Triple Isotope Composition of Dissolved Oxygen  

Microsoft Academic Search

Although knowledge of accurate Primary Productivity (PPr) rates is essential to the understanding of ocean carbon cycling, the standard method of determining ocean productivity, 14C labeling, often yields uncertain results. Typically, 14C-derived PPr rates fall ambiguously between gross and net productivity because the method is sensitive to recycling of a relatively small POC pool. Bottle incubations using labeled oxygen produced

L. W. Juranek; P. D. Quay; D. M. Karl



Stimulating carbon efficient supply chains : carbon labels and voluntary public private partnerships  

E-print Network

This thesis looks at the potential of labeling products with life cycle greenhouse gas emission information as a bottom-up, complementary alternative to carbon cap and trade systems. By improving the transparency of product ...

Tan, Kwan Chong



Integration of High Accuracy N-Terminus Identification in Peptide Sequencing and Comparative Protein Analysis Via Isothiocyanate-Based Isotope Labeling Reagent with ESI Ion-trap TOF MS  

Microsoft Academic Search

A multifunctional isothiocyanate-based isotope labeling reagent, [d\\u000a 0]-\\/[d\\u000a 6]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide\\u000a sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling\\u000a reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement,\\u000a and facilitating peptide sequencing. More significantly, DMPITC-based labeling

Jiapeng Leng; Haoyang Wang; Li Zhang; Jing Zhang; Hang Wang; Tingting Cai; Jinting Yao; Yinlong Guo



Development of isotope labeling liquid chromatography mass spectrometry for mouse urine metabolomics: quantitative metabolomic study of transgenic mice related to Alzheimer's disease.  


Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC-MS) for relative quantification of over 950 putative metabolites using 20 ?L of urine as the starting material. The workflow involves aliquoting 10 ?L of an individual urine sample for (12)C-dansylation labeling that target amines and phenols. Another 10 ?L of aliquot was taken from each sample to generate a pooled sample that was subjected to (13)C-dansylation labeling. The (12)C-labeled individual sample was mixed with an equal volume of the (13)C-labeled pooled sample. The mixture was then analyzed by LC-MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC-MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer's disease (FAD) throughout the course of their pathological deposition of beta amyloid (A?). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15-17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous metabolomics studies in human cerebrospinal fluid or blood samples. This work illustrates the utility of this isotope labeling LC-MS method for biomarker discovery using mouse urine metabolomics. PMID:25164377

Peng, Jun; Guo, Kevin; Xia, Jianguo; Zhou, Jianjun; Yang, Jing; Westaway, David; Wishart, David S; Li, Liang



Isolation, In-111 labeling, and abscess detection efficiency of rabbit polymorphonuclear leukocytes (PMN) from blood and peritoneal fluid  

SciTech Connect

In-111 labeled blood and peritoneal exudate PMN were compared for labeling efficiency and ability to migrate to sites of experimental abscesses using both direct sampling and visual imaging techniques. Blood PMN were prepared by combining heparinized blood with 6% Hetastarch for 1 hour and layering the plasma over a double density Ficoll-Hy-paque gradient (S.G. 1.076 over 1.141). The PMN layer (90-99% PMN) at the interface yielded 10/sup 6/-10/sup 7/ PMN from 80-120 ml of blood. Peritoneal PMN were obtained by infusion of 0.1% glycogen, followed by infusion of saline after 4 or 18 hours. The exudate yielded 10/sup 7/-10/sup 8/ PMN (80-99% PMN). PMN suspensions were labeled for 30 minutes by addition of 100 of In-111-oxine, then washed twice. Percent cell-associated radioactivity of the labeled blood, 4 hour, and 18 hour peritoneal PMN was 89%, 88%, and 86%. The labeled PMN were injected intravenously into rabbits which had two of three abdominal capsules (table tennis balls drilled with 250 1.5 mm holes) inoculated with Staphylococcus aureus 4 hours earlier. Peak venous recovery of circulating labeled PMN, for blood, 4 hour and 18 hour peritoneal PMN was 60%, 43%, and 19%. Gamma camera images 24 hours after infusion into infected rabbits were superior with 4 hour peritoneal PMN. The peritoneal PMN harvested 4 hours after glycogen stimulation are simple to prepare, are obtainable in greater numbers than blood PMN, and result in better abscess visualization.

Bettin, K.M.; Elson, M.K.; Gerding, D.N.; Bamberger, D.M.; Forstrom, L.A.; Shafer, R.B.



Highly efficient fluorescent interstrand photo-crosslinking of DNA duplexes labeled with 5-fluoro-4-thio-2'-o-methyluridine.  


The formation of a fluorescent photoadduct between 5-fluoro-4-thiouridine ((FS) U), in the sequence context 5'-A(FS) UA-3' and incorporated into a synthetic oligonucleotide either at its 3'- or 5'-end, and one of the thymines of the TAT motif in a complementary target DNA strand led to photo-crosslinking of the two strands for several oligonucleotide constructs. Enzymatic digestion, MS, UV, and fluorescence spectral analyses of the interstrand crosslinked oligonucleotides revealed the identity of the thymidine that participates in the photo-crosslinking reaction as well as the diastereomeric structures of the crosslinks. The proposed pathways of interstrand photo-crosslinking are supported by experiments with isotopically labeled oligonucleotide constructs and visualized by means of molecular dynamics simulations. PMID:25111776

Nowak-Karnowska, Joanna; Chebib, Ziad; Milecki, Jan; Franzen, Stefan; Skalski, Bohdan



Importance of bacterivory and preferential selection toward diatoms in larvae of Crepidula fornicata (L.) assessed by a dual stable isotope (13C, 15N) labeling approach  

NASA Astrophysics Data System (ADS)

In Europe, the gastropod Crepidula fornicata is an invasive species characterized by a long reproductive period (from February to November). Thus, its larvae are exposed to variations in available food sources (in terms of quantity and quality). We aimed to investigate if bacteria could contribute to larval food both in presence or absence of phytoplankton, and to compare these results to seasonal variations of bacteria and phytoplankton abundances at a coastal site in the English Channel. First, ingestion of fluorescent beads of 0.5 to 2 ?m diameter, showed that larvae were able to ingest particles of typical bacterial size. Then we used a dual stable isotope labeling approach which consisted in labeling a bacterial pelagic community with 15N and a diatom (Chaetoceros gracilis) culture with 13C, and supplying larvae with 15N-labeled bacteria, 13C-labeled diatoms, and both labeled sources. This technique has, to our knowledge, never been applied to invertebrate larvae. After 24 h of experiment, larvae were significantly enriched in all treatments: + 21.5‰ (??13C) when supplied with diatoms, + 1364‰ (??15N) when supplied with bacteria, and + 24‰ (??13C) and + 135‰ (??15N) when supplied with the two mixed sources. These results indicated that bacteria can contribute to the larval nutrition in C. fornicata, even in the presence of phytoplankton. Our results however suggested that larvae of C. fornicata preferentially used diatoms and showed that the supply of free bacteria did not alter the uptake of diatoms. Considering the seasonal variations of bacteria and phytoplankton abundances at the study site, these results suggested that bacteria may constitute a complementary resource for the larvae of C. fornicata when phytoplankton is abundant and may become a substitute resource when phytoplankton is less available. This approach offers promising perspectives to trace food sources and assess nitrogen and carbon fluxes between planktotrophic larvae and their preys.

Leroy, Fanny; Riera, Pascal; Jeanthon, Christian; Edmond, Frédérique; Leroux, Cédric; Comtet, Thierry



[Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].  


The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium ?. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 ? 2 O and 2% [ 2 ?]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 ? 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 ? 2 O followed by subsequent selection of separate colonies stable to the action of 2 ? 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 ? 2 O in growth media, from 17% (on growth medium with 24,5% 2 ? 2 O) up to 75% (on growth medium with 98% 2 ? 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions. PMID:25249528

Mosin, O V; Shvets, V I; Skladnev, D A; Ignatov, I



Development of a routine method for the simultaneous confirmation and determination of clenbuterol in urine by minimal labeling isotope pattern deconvolution and GC-EI-MS.  


A novel and fast routine method for the simultaneous determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry (GC-EI-MS) has been developed. The method employs isotope dilution mass spectrometry (IDMS) and is based on a combination of minimal labeling (a single (13)C label in the molecule) and isotope pattern deconvolution (IPD). This new methodology does not require the construction of a methodological calibration graph, and was compared with the classical IDMS procedure employed in clenbuterol analysis based on the use of a deuterated compound as internal standard (d(9)-clenbuterol) and a calibration curve. The sample preparation consists of simple extraction with dichloromethane, which was dried and derivatized with chloro(chloromethyl)dimethylsilane, generating a cyclic dimethylsilamorpholine (DMS) derivative suitable for GC(EI)MS detection and identification. This compound produces five intense ions in the electron ionization source, which allow the presence of clenbuterol to be confirmed in just one analysis, as demanded by European Union directives. The accuracy of the method was studied by performing recovery experiments at different concentration levels (from 0.3 to 5 ng g(-1)) in 5 mL bovine urine samples using two labeled compounds: an in-house-synthesized (13)C(1)-clenbuterol and a commercially available d(9)-clenbuterol. The detection limit of the method in human urine was 0.050 ng g(-1) with a sample volume of 10 mL, and is thus suitable for antidoping control purposes. Finally, the (13)C(1)-clenbuterol standard was employed for the determination of clenbuterol in two reference materials, BCR-503 and BCR-504 (lyophilized bovine urine). The concentrations obtained were in agreement with the certified values, with a reproducibility of below 1% RSD. PMID:22241580

González-Antuña, Ana; Rodríguez-González, Pablo; Lavandera, Iván; Centineo, Giuseppe; Gotor, Vicente; García Alonso, J Ignacio



Combined Chemical and Enzymatic Stable Isotope Labeling for Quantitative Profiling of Detergent-insoluble Membrane Proteins Isolated Using Triton X-100 and Brij-96  

PubMed Central

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five non-cysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content. PMID:16457601

Blonder, Josip; Yu, Li-Rong; Radeva, Galina; Chan, King C.; Lucas, David A.; Waybright, Timothy J.; Issaq, Haleem J.; Sharom, Frances J.; Veenstra, Timothy D.



Isotope-Coded and Affinity-Tagged Cross-Linking (ICATXL): An Efficient Strategy to Probe Protein Interaction Surfaces  

E-print Network

Isotope-Coded and Affinity-Tagged Cross-Linking (ICATXL): An Efficient Strategy to Probe Protein; E-mail: Protein bait affinity studies on the yeast proteome have begun to reveal of the cross-linked products sought. Incorporation of affinity tags into cross-linkers can reduce

Craik, Charles S.


Efficient access to the non-reducing end of low molecular weight heparin for fluorescent labeling.  


A novel thiol fluorophore was synthesized to be selectively attached to the non-reducing end of low molecular weight heparin (LMWH) via a Michael addition. Double labeling of LMWH was demonstrated to be a feasible approach for the determination of heparinase II activity by FRET. PMID:24846618

Wang, Zongqiang; Shi, Chen; Wu, Xuri; Chen, Yijun



Influences of Gas Stream Conditions on Efficiency of Tritiated Moisture Collection with P2O5Desiccant and Isotope Effect  

Microsoft Academic Search

A method was proposed previously for collection and measurement of tritiated moisture in gas stream using P2O5-desiccant. Influences of the gas humidity, the gas flow rate and the distance between gas nozzle and P2O5-desiccant layer surface on the moisture collection efficiency have been examined through experiments, and the isotope effect on the collection has been investigated. The collection efficiency is

Kenji KOTOH; Katsuya MIURA; Yousuke KASHIO; Masabumi NISHIKAWA



Time-shared experiments for efficient assignment of triple-selectively labeled proteins  

NASA Astrophysics Data System (ADS)

Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of 13C 90° pulses allows to either select or discriminate against 13C? or 13C? spins coupled to 15N. As a result, 1H-15N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns.

Löhr, Frank; Laguerre, Aisha; Bock, Christoph; Reckel, Sina; Connolly, Peter J.; Abdul-Manan, Norzehan; Tumulka, Franz; Abele, Rupert; Moore, Jonathan M.; Dötsch, Volker



Parallel ?-sheet vibrational couplings revealed by 2D IR spectroscopy of an isotopically labeled macrocycle: Quantitative benchmark for the interpretation of amyloid and protein infrared spectra  

PubMed Central

Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel ?-sheets. Using a synthetic macrocycle that enforces a two stranded parallel ?-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly 13C=18O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequen cies of the labels ranged from 1585 to 1595 cm?1, with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the linewidths and frequencies nearly quantitatively. Historically, the characteristic features of ?-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel ?-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm?1 linewidth. Narrower linewidths indicate that the amide I backbone is solvent protected inside the macrocycle. This work provides calculated and experimentally verified couplings for parallel ?-sheets that can be used in structure-based models to simulate and interpret the infrared spectra of ?-sheet containing proteins and protein assemblies, such as amyloid fibers. PMID:23113791

Woys, Ann Marie; Almeida, Aaron M.; Wang, Lu; Chiu, Chi Cheng; McGovern, Michael; de Pablo, Juan J.; Skinner, James L.; Gellman, Samuel H.; Zanni, Martin T.



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


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

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



Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry  

E-print Network

Sciences, Chinese Academy of Sciences, Shanghai, China Abstract The biosynthesis of neuroendocrine peptidesExamination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d10

Tian, Weidong


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

SciTech Connect

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

Hines, J.V.; Landry, S.M.; Varani, G.; Tinoco, I. Jr. (Univ. of California, Berkeley, CA (United States) Lawrence Berkeley Lab., CA (United States))



Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling1[C][W  

PubMed Central

In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling. PMID:23085839

Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian; Durner, Jorg



Using stable isotopes to reconcile differences in nitrogen uptake efficiency relative to late season fertilization of northern red oak seedlings in Wisconsin bare-root nurseries  

NASA Astrophysics Data System (ADS)

Cultural applications (e.g., timing, amount) of nitrogen (N) fertilizer in bareroot tree nurseries have been assessed for some time. However, the use of different metrologies to quantify the efficient use of fertilizer N and its allocation within biomass has confounded comparisons between fertilization regimes. This inconsistency is especially problematic when quantifying N fertilizer uptake efficiency (NFUE) of late season N fertilization in northern red oak (Quercus rubra L.) (NRO) seedlings characterized by episodic flushes in growth and N storage in perennial tissue to support spring growth. The use of isotopic tracers could help elucidate these differences. We therefore hypothesized that: 1) calculations of NFUE using isotopically enriched fertilizer would yield lower, more precise estimates of NFUE relative to traditional methods due to differences in the accounting of mineralized and reabsorbed N, and 2) a significant fraction of leaf N in older leaves (early flushes) would be reabsorbed into root and shoot tissue before abscission relative to leaves produced toward the end of the growing season (late flushes). To test these hypotheses, we conducted an experiment in two-year old NRO seedlings at two bare-root nurseries in Wisconsin. We applied a total of 147 mg N seedling-1 in pulses from early July after the seedlings completed their second leaf flush until late August. The treatments consisted of three replicated plots of 15N enriched (1.000 atom%) ammonium sulfate, three non-enriched plots, and three unfertilized plots (controls) at each nursery. Subsequent changes in plant N uptake and N allocation were quantified from destructively harvested samples taken at 40, 60, and 120 days after the fertilization began. We evaluated three common methods currently used to estimate NFUE (total N without control, total N with control, and isotopic difference). The total N without control method overestimated mean NFUE by 3.2 times relative to the isotope method, because mineralized N uptake and reabsorption of leaf N was unaccounted for. The total N with control method also overestimated mean NFUE, but only by 20% relative to the isotope method; variation associated with the effects of N fertilization on mineralization and immobilization was large enough to preclude significant difference between these methods. The difference of non-labeled N between day 60 and day 120 revealed that the roots and shoots absorbed 95% and 5%, respectively, of initial leaf N. However, isotopic mass balance between day 60 and day 120 indicated that the NRO seedlings did not reabsorb leaf fertilized N from the youngest leaves before abscission. This study shows that using stable isotopes to understand plant-soil interactions in response to fertilization will help elucidate the contribution of additional N fluxes (e.g., N reabsorption) within perennial plants and thus improve fertility management of production systems.

Fujinuma, R.; Balster, N. J.



Combined Gel Probe and Isotope Labeling Technique for Measuring Dissimilatory Nitrate Reduction to Ammonium in Sediments at Millimeter-Level Resolution?  

PubMed Central

Dissimilatory NO3? reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO3? concentrations yields potential rather than actual activities of dissimilatory NO3? reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO3? consumption. The water column of the sediment core is amended with 15NO3? at the in situ 14NO3? concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH4+ dissolved in the gel slices is chemically converted by hypobromite to N2 in reaction vials. The isotopic composition of N2 is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of 15NH4+. The results were compared to the NH4+ microsensor profiles measured in freshwater sediment and quartz sand and to the N2O microsensor profiles measured in acetylene-amended sediments to trace denitrification. PMID:20656865

Stief, Peter; Behrendt, Anna; Lavik, Gaute; De Beer, Dirk



A Method Revealing Bacterial Cell-wall Architecture by Time-dependent Isotope Labeling and Quantitative Liquid Chromatography/Mass Spectrometry  

PubMed Central

The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The results allow us to track the fate of cell-wall precursors (which contain the vancomycin-binding site) in Enterococcus faecium, a leading antibiotic-resistant pathogen. By comparing isotopic enrichments of post-insertionally modified cell-wall precursors, we find that tripeptides and species without Asx bridges are specific to mature cell wall. Additionally, we find that the sequence of cell-wall maturation varies throughout a cell cycle. We suggest that actively dividing E. faecium cells have three zones of unique peptidoglycan processing. Our results reveal new organizational characteristics of the bacterial cell wall that are important to understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens. PMID:19281243

Patti, Gary J.; Chen, Jiawei; Gross, Michael L.



Regioselective synthesis of isotopically labeled ?9-tetrahydrocannabinolic acid A (THCA-A-D3) by reaction of ?9-tetrahydrocannabinol-D3 with magnesium methyl carbonate.  


For the reliable quantification of ?9-tetrahydrocannabinolic acid A (THCA-A), the biogenetic precursor of ?9-tetrahydrocannabinol (THC), in biological matrices by LC-MS/MS and GC-MS(/MS), an isotopically labeled internal standard was synthesized starting from ?9-tetrahydrocannabinol-D(3) (THC-D(3)). Synthesis strategy was based on a method reported by Mechoulam et al. in 1969 using magnesium methyl carbonate (MMC) as carboxylation reagent for the synthesis of cannabinoid acids. Preliminary experiments with THC to optimize yield of the product (THCA-A) resulted in the synthesis of the positional isomer tetrahydrocannabinolic acid B (THCA-B) as a byproduct. Using the optimized conditions for the desired isomer, THCA-A-D(3) was prepared and isolated with a yield of approx. 10% after two synthesis cycles. Isotope purity was estimated to be >99% by relative abundance of the molecular ions. The synthesized compound proved to be suitable as an internal standard for quantification of THCA-A in serum and hair samples of cannabis consumers. PMID:22921834

Roth, Nadine; Wohlfarth, Ariane; Müller, Michael; Auwärter, Volker



The effect of FISH and CARD-FISH on the isotopic composition of (13)C- and (15)N-labeled Pseudomonas putida cells measured by nanoSIMS.  


The use of nanoSIMS for the exploration of microbial activities in natural habitats often implies that stable isotope tracer experiments are combined with in situ hybridization techniques (i.e. fluorescence in situ hybridization (FISH) or catalyzed reporter deposition (CARD)-FISH). In this study, Pseudomonas putida grown on (13)C- and (15)N-labeled carbon and nitrogen, collected in exponential growth and stationary phases, was hybridized and analyzed by nanoSIMS. It was shown that (13)C and (15)N fractions decreased after FISH and CARD-FISH in comparison to chemically untreated cells. However, the fractions were influenced differently by various treatments. After paraformaldehyde fixation of exponentially growing cells, a reduction of the (13)C and (15)N fractions was measured from 94±1.2% and 89.5±3.8% to 90.2±0.8% and 64±4.6%, respectively, indicating that nitrogen isotopic composition was most influenced. A further decrease of the (13)C and (15)N fractions to 80.7±6.5 and 59.5±4.1%, respectively, was measured after FISH, while CARD-FISH decreased the fractions to 57.4±3.0% and 47.1±4.1%, respectively. The analysis of cells collected in different growth phases revealed that the effect of various treatments seemed to be dependent on the cell's physiological state. In addition, a mathematical model that can be used in further studies was developed in order to calculate the amount of carbon introduced into the cells by chemical treatments. These results can be valuable for environmental FISH-nanoSIMS studies where the isotopic composition of single cells will be used to quantitatively assess the importance of specific populations to certain biochemical processes and determine budget estimations. PMID:24702905

Musat, Niculina; Stryhanyuk, Hryhoriy; Bombach, Petra; Adrian, Lorenz; Audinot, Jean-Nicolas; Richnow, Hans H



Iron uptake and ferrokinetics in healthy male subjects of an iron-based oral phosphate binder (SBR759) labeled with the stable isotope (58)Fe.  


SBR759 is a novel polynuclear iron(iii) oxide-hydroxide starch·sucrose·carbonate complex being developed for oral use in chronic kidney disease (CKD) patients with hyperphosphatemia on hemodialysis. SBR759 binds inorganic phosphate released by food uptake and digestion in the gastro-intestinal tract increasing the fecal excretion of phosphate with concomitant reduction of serum phosphate concentrations. Considering the high content of ?20% w/w covalently bound iron in SBR759 and expected chronic administration to patients, absorption of small amounts of iron released from the drug substance could result in potential iron overload and toxicity. In a mechanistic iron uptake study, 12 healthy male subjects (receiving comparable low phosphorus-containing meal typical for CKD patients: ?1000 mg phosphate per day) were treated with 12 g (divided in 3 × 4 g) of stable (58)Fe isotope-labeled SBR759. The ferrokinetics of [(58)Fe]SBR759-related total iron was followed in blood (over 3 weeks) and in plasma (over 26 hours) by analyzing with high precision the isotope ratios of the natural iron isotopes (58)Fe, (57)Fe, (56)Fe and (54)Fe by multi-collector inductively coupled mass spectrometry (MC-ICP-MS). Three weeks following dosing, the subjects cumulatively absorbed on average 7.8 ± 3.2 mg (3.8-13.9 mg) iron corresponding to 0.30 ± 0.12% (0.15-0.54%) SBR759-related iron which amounts to approx. 5-fold the basal daily iron absorption of 1-2 mg in humans. SBR759 was well-tolerated and there was no serious adverse event and no clinically significant changes in the iron indices hemoglobin, hematocrit, ferritin concentration and transferrin saturation. PMID:25017110

Gschwind, Hans-Peter; Schmid, Dietmar G; von Blanckenburg, Friedhelm; Oelze, Marcus; van Zuilen, Kirsten; Slade, Alan J; Stitah, Sylvie; Kaufmann, Daniel; Swart, Piet



Water use efficiency and carbon isotope composition of plants in a cold desert environment.  


The effects of the availabilities of water and nitrogen on water use efficiency (WUE) of plants were investigated in a sagebrush steppe. The four species studied wereArtemisia tridentata (shrub),Ceratoides lanata (suffrutescent shrub),Elymus lanceolatus (rhizomatous grass), andElymus elymoides (tussock grass). Water and nitrogen levels were manipulated in a two-by-two factorial design resulting in four treatments: control (no additions), added water, added nitrogen, and added water and nitrogen. One instantaneous and two long-term indicators of WUE were used to testa priori predictions of the ranking of WUE among treatments. The short-term indicator was the instantaneous ratio of assimilation to transpiration (A/E). The long-term measures were 1) the slope of the relationship between conductance to water vapor and maximum assimilation and 2) the carbon isotope composition (?(13)C) of plant material. Additional water decreased WUE, whereas additional nitrogen increased WUE. For both A/E and ?(13)C, the mean for added nitrogen alone was significantly greater than the mean for added water alone, and means for the control and added water and nitrogen fell in between. This ranking of WUE supported the hypothesis that both water and nitrogen limit plant gas exchange in this semiarid environment. The short- and long-term indicators were in agreement, providing evidence in support of theoretical models concerning the water cost of carbon assimilation. PMID:23494339

Toft, N L; Anderson, J E; Nowak, R S



Quantitation of methadone enantiomers in humans using stable isotope-labeled (2H3)-, (2H5)-, and (2H8)Methadone  

SciTech Connect

A new technique for simultaneous stereoselective kinetic studies of methadone enantiomers was developed using three deuterium-labeled forms of methadone and GLC-chemical-ionization mass spectrometry. A racemic mixture (1:1) of (R)-(-)-(2H5)methadone (l-form) and (S)-(R)-(2H3)methadone (d-form) was administered orally in place of a single daily dose of unlabeled (+/-)-(2H0)methadone in long-term maintenance patients. Racemic (+/-)-(2H8)methadone was used as an internal standard for the simultaneous quantitation of (2H0)-, (2H3)-, and (2H5)methadone in plasma and urine. A newly developed extraction procedure, using a short, disposable C18 reversed-phase cartridge and improved chemical-ionization procedures employing ammonia gas, resulted in significant reduction of the background impurities contributing to the ions used for isotopic abundance measurements. These improvements enabled the measurement of labeled plasma methadone levels for 120 hr following a single dose. This methodology was applied to the study of methadone kinetics in two patients; in both patients, the analgesically active l-enantiomer of the drug had a longer plasma elimination half-life and a smaller area under the plasma disappearance curve than did the inactive d-form.

Nakamura, K.; Hachey, D.L.; Kreek, M.J.; Irving, C.S.; Klein, P.D.



Isotopic labeling studies of the mechanism of dehydrogenation of methanol to methyl formate over copper-based catalysts  

Microsoft Academic Search

Deuterium labeling has been used to study the processes occurring during the conversion of methanol to methyl formate over copper catalysts at 180-210°C and pressures of 0.3 to 1 atm. Deuterium substitution has a dramatic effect on rate, which decreases in the ratio 8:4:2:1 in the series CHâOH, CHâOD, CDâOH, CDâOD. This can be interpreted as the product of a

N. W. Cant; S. P. Tonner; D. L. Trimm; M. S. Wainwright



Proteins with high turnover rate in barley leaves estimated by proteome analysis combined with in planta isotope labeling.  


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 (15)N 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 (15)N 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 (14)N-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 (15)N 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 (15)N 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

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



Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using their fully 15N-labeled analogues as internal standards.  


Oxidatively induced DNA damage is implicated in disease, unless it is repaired by DNA repair. Defects in DNA repair capacity may be a risk factor for various disease processes. Thus, DNA repair proteins may be used as early detection and therapeutic biomarkers in cancer and other diseases. For this purpose, the measurement of the expression level of these proteins in vivo will be necessary. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS) for the identification and quantification of DNA repair proteins human 8-hydroxyguanine-DNA glycosylase (hOGG1) and Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which are involved in base-excision repair of oxidatively induced DNA damage. We overproduced and purified (15)N-labeled analogues of these proteins to be used as suitable internal standards to ensure the accuracy of quantification. Unlabeled and (15)N-labeled proteins were digested with trypsin and analyzed by LC-MS/MS. Numerous tryptic peptides of both proteins were identified on the basis of their full-scan mass spectra. These peptides matched the theoretical peptide fragments expected from trypsin digestion and provided statistically significant protein scores that would unequivocally identify these proteins. We also recorded the product ion spectra of the tryptic peptides and defined the characteristic product ions. Mixtures of the analyte proteins and their (15)N-labeled analogues were analyzed by selected-reaction monitoring on the basis of product ions. The results obtained suggest that the methodology developed would be highly suitable for the positive identification and accurate quantification of DNA repair proteins in vivo as potential biomarkers for cancer and other diseases. PMID:21619077

Dizdaroglu, Miral; Reddy, Prasad T; Jaruga, Pawel



Efficient 18F-Labeling of Large 37-Amino Acid pHLIP Peptide Analogues and their Biological Evaluation  

PubMed Central

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP®) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pHe<7). Labeling of peptides with [18F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known “click” methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC “click chemistry” for the simple and efficient 18F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and a L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[18F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ? 98%. The subsequent CuI catalyzed “click” reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [18F]-D-WT-pHLIP and [18F]-L-K-pHLIP were obtained with total radiochemical yields of 5–20% after HPLC purification. The total reaction time was only 85 min including formulation. In vitro stability tests revealed high stability of the [18F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65 and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [18F]-D-WT-pHLIP and the negative control [18F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the 18F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [18F]-pHLIP analogues as potential PET tracers. PMID:22784215

Daumar, Pierre; Wanger-Baumann, Cindy A.; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D.; Andreev, Oleg A.; Reshetnyak, Yana K.; Lewis, Jason S.



Comparing Isotope Dilution Methods to Label Free Quantitation Methods For The Analysis of Vaccine Standards and Products  

PubMed Central

Determining protein content in biologics is an important part of the production process. An example of interest to public health is the influenza vaccine, where the amount of the major antigenic protein hemagglutinin and the amount of egg proteins from the expression system are regulated. Mass spectrometry has advantages of higher specificity, speed and permits other proteins to be analyzed simultaneously. Here, we present the use of a MRM method for quantitation of hemagglutinin and other vaccine proteins developed in our laboratory and compare this approach to a label free method (MSE) for simultaneous identification and absolute quantitation of virus and egg proteins. Influenza vaccine samples were tryptically digested using a protocol developed in our laboratory to ensure consistent and reproducible results. Traditional IDMS measurements were made on ThermoFisher Scientific TSQ quantum triple quadrupole platform. Label free methods (LC-MSE) were performed on a Waters qTOF Premier platform and the PLGS software. Both instruments were coupled to an Identical Agilent 1200 LC platform to insure an accurate comparison. The results of the study illustrated that IDMS remains the gold standard for absolute protein quantitation via mass spectrometry. MSE performed with comparable precision and accuracy in cases where the sample was less complex (monovalent pandemic vaccines vs. seasonal trivalent vaccines). In addition the choice of peptides made by the MSE algorithm and the choice of influenza proteins used in the database also affected the precision and accuracy of the MSE absolute quantitation results.

Winne, Emily; Santana, Wanda I.; Williams, Tracie L.; Barr, John R.; Bundy, Jonathan




EPA Science Inventory

The purpose of the project is to reduce pollution and environmental degradation by increasing the efficiency of energy end-uses in the industrial and household sectors of key Asian and Latin American countries. This will be accomplished by encouraging the adoption and harmo...


Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells  

PubMed Central

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and [Ca2+]i between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro. PMID:25234466

Zhang, Ruiping; Li, Jing; Li, Jianding; Xie, Jun



Binding of ?4?5 by Adenosine A1 and A2A Receptors Determined by Stable Isotope Labeling with Amino Acids in Cell Culture and Mass Spectrometry†  

PubMed Central

Characterization of G protein ?? dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity or availability. As a new approach, we used quantitative mass spectrometry to characterize native ?? dimers associated with adenosine A1:?i1 and adenosine A2A:?S receptor fusion proteins expressed in HEK-293 cells. Cells expressing A1:?i1 were cultured in media containing [13C6] Arg and [13C6] Lys, and ?? labeled with heavy isotopes purified. Heavy ?? was combined with either recombinant ?? purified from Sf9 cells, ?? purified from the A2A:?S expressed in HEK-293 cells cultured in standard media, or an enriched ?? fraction from HEK-293 cells. Samples were separated by SDS-PAGE, and protein bands containing ? and ? were excised, digested with trypsin, separated by HPLC and isotope ratios analyzed by mass spectrometry. Three ? isoforms, ?1, ?2 and ?4, and seven ? isoforms, ?2, ?4, ?5, ?7, ?10, ?11 and ?12 were identified in the analysis. ?1 and ?5 were most abundant in the enriched ?? fraction, and this ?? profile was generally mirrored in the fusion proteins. However, both A2A:?S and A1:?i1 bound more ?4 and ?5 compared to the enriched ?? fraction; also, more ?4 was associated with A2A:?S than A1:?i1. Both fusion proteins also contained less ?2, ?10 and ?12 than the enriched ?? fraction. These results suggest that preferences for particular ?? isoforms may be driven in part by structural motifs common to adenosine receptor family members. PMID:21128647

Wang, Dora Bigler; Sherman, Nicholas E.; Shannon, John D.; Leonhardt, Susan A.; Mayeenuddin, Linnia H.; Yeager, Mark; McIntire, William E.



Microwave-assisted (18) O labeling of Fmoc-protected amino acids.  


Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of (18) O-labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave-assisted procedure. This approach resulted in reduction of reaction time to 15?min and increased reaction efficiency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. PMID:25098748

Modzel, Maciej; P?óciennik, Halina; Kluczyk, Alicja; Stefanowicz, Piotr; Szewczuk, Zbigniew



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


A method for the preparation of multi-gramme quantities of N-methyl-d3 -N-nitroso-p-toluenesulfonamide (Diazald-d3 ) and N-methyl-(13) C-N-nitroso-p-toluenesulfonamide (Diazald-(13) C) and their conversion to diazomethane-d2 and diazomethane-(13) C, respectively, is presented. This approach uses robust and reliable chemistry, and critically, employs readily commercially available and inexpensive methanol as the label source. Several reactions of labelled diazomethane are also reported, including alkene cyclopropanation, phenol methylation and ?-diazoketone formation, as well as deuterium scrambling in the preparation of diazomethane-d2 and subsequent methyl esterification of benzoic acid. PMID:25318972

Shields, Samuel W J; Manthorpe, Jeffrey M



Intraoperative beta probe: A device for detecting tissue labeled with positron or electron emitting isotopes during surgery  

SciTech Connect

An intraoperative beta probe was designed, built, and tested for detection of radio-labeled malignant tissues that has the advantage of being selectively sensitive to beta while insensitive to gamma radiation. Since beta radiation (electrons or positrons) has a short range in tissue, this probe is ideal for detecting tracers in tumors at the surface of the surgical field. This probe contains a plastic scintillation detector sensitive to beta rays and to a lesser degree some background gamma rays. A second detector counts spurious gamma rays and allows for their subtraction from the activity measured by the first detector. Sensitivity of the dual probe for I-131 and F-18 was measured to be 108 counts/s/kBq (4000 counts/s/[mu]Ci). The dual-detector probe faithfully measured the 10:1 tumor'' to background ratio of radioactivity concentrations in a simulated environment of a tumor in the presence of intense background 511 keV photons. In another phantom experiment, simulating abdominal tumor deposits with various realistic I-131 radioactive concentrations, the probe was able to accurately identify tumors of approximately 50 mg with a tumor/normal radioactivity concentration of 3/1 in 10 s.

Daghighian, F. (Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 (United States)); Mazziotta, J.C. (Division of Brain Mapping of Neuropsychiatric Institute, Department of Neurology, Department of Pharmacology, Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California 90024 (United States)); Hoffman, E.J. (Department of Pharmacology, Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California 90024 (United States)); Shenderov, P.; Eshaghian, B. (Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 (United States)); Siegel, S.; Phelps, M.E. (Department of Pharmacology, Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California 90024 (United States))



Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen.

Svensson, Jan, E-mail: [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden) [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Bergman, Ann-Charlotte [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden)] [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Adamson, Ulf [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)] [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Blombaeck, Margareta [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden)] [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Wallen, Hakan; Joerneskog, Gun [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)] [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)



Identifying Natural Substrates for Dipeptidyl Peptidases 8 and 9 Using Terminal Amine Isotopic Labeling of Substrates (TAILS) Reveals in Vivo Roles in Cellular Homeostasis and Energy Metabolism*?  

PubMed Central

Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease. PMID:23519473

Wilson, Claire H.; Indarto, Dono; Doucet, Alain; Pogson, Lisa D.; Pitman, Melissa R.; McNicholas, Kym; Menz, R. Ian; Overall, Christopher M.; Abbott, Catherine A.



Identifying natural substrates for dipeptidyl peptidases 8 and 9 using terminal amine isotopic labeling of substrates (TAILS) reveals in vivo roles in cellular homeostasis and energy metabolism.  


Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease. PMID:23519473

Wilson, Claire H; Indarto, Dono; Doucet, Alain; Pogson, Lisa D; Pitman, Melissa R; McNicholas, Kym; Menz, R Ian; Overall, Christopher M; Abbott, Catherine A



Novel tracer method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies.  


Gaseous nitrous acid (HONO), the protonated form of nitrite, contributes up to ?60% to the primary formation of hydroxyl radical (OH), which is a key oxidant in the degradation of most air pollutants. Field measurements and modeling studies indicate a large unknown source of HONO during daytime. Here, we developed a new tracer method based on gas-phase stripping-derivatization coupled to liquid chromatography-mass spectrometry (LC-MS) to measure the 15N relative exceedance, ?(15N), of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye, purified by solid phase extraction (SPE), and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In the optimal working range of ?(15N)=0.2-0.5, the relative standard deviation of ?(15N) is <4%. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method was applied to measure HO15NO emissions from soil in a dynamic chamber with and without spiking 15) labeled urea. The identification of HO15NO from soil with 15N urea addition confirmed biogenic emissions of HONO from soil. The method enables a new approach of studying the formation pathways of HONO and its role for atmospheric chemistry (e.g., ozone formation) and environmental tracer studies on the formation and conversion of gaseous HONO or aqueous NO2- as part of the biogeochemical nitrogen cycle, e.g., in the investigation of fertilization effects on soil HONO emissions and microbiological conversion of NO2- in the hydrosphere. PMID:24954648

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



Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer’s ?-amyloid peptide for NMR-based structural analysis  

PubMed Central

Amyloid fibrils of Alzheimer’s ?-amyloid peptide (A?) are a primary component of amyloid plaques, a hallmark of Alzheimer’s disease (AD). Enormous attention has been given to the structural features and functions of A? in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue A?(1–40), the most abundant A? in brains, for structural studies by NMR spectroscopy. Over-expression of A?(1–40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR?) in E. Coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-A? fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ~7 mg and ~1.5 mg of unlabeled A?(1–40) and uniformly 15N- and/or 13C-protein A?(1–40) from 1 L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled A? and 1H/15N HSQC solution NMR spectrum of the obtained 15N-labeled A? in the monomeric form confirmed the expression of native A?(1–40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified A?(1–40) self-assembles into ?-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant A?(1–40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are 13C- and 15N-labeled for NMR experiments. PMID:21640828

Long, Fei; Cho, Wonhwa; Ishii, Yoshitaka



Efficient sampling of early signal arrival for estimation of perfusion and transit time in whole-brain arterial spin labeling.  


Arterial spin labeling can be used to measure both cerebral perfusion and arterial transit time. However, accurate estimation of these parameters requires adequate temporal sampling of the arterial spin labeling difference signal. In whole-brain multislice acquisitions, two factors reduce the accuracy of the parameter estimates: saturation of labeled blood in transit and inadequate sampling of early difference signal in superior slices. Label saturation arises when slices are acquired inferior-to-superior such that slice selection in proximal slices spoils the label for a distal slice. Inadequate sampling arises when the time spent acquiring inferior slices is too long to allow early sampling of the difference signal in superior slices. A novel approach to multislice imaging is proposed to address these two issues. In round-robin arterial spin labeling, slices are acquired in a different order after every pair of control-label acquisitions. Round-robin arterial spin labeling enables the acquisitions of all slices across the same range of postlabel delays in a descending superior-to-inferior order. This eliminates the temporal sampling problem and greatly reduces label saturation. Arterial transit time estimates obtained for the whole brain with round-robin arterial spin labeling show better agreement with a single-slice acquisition than do conventional multislice acquisitions. PMID:22189961

Lee, Wayne; Janik, Rafal; Scouten, Amy; Stefanovic, Bojana; Sled, John G



The solution structure refinement of the paramagnetic reduced high-potential iron-sulfur protein I from Ectothiorhodospira halophila by using stable isotope labeling and nuclear relaxation.  


The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons. PMID:8917441

Bertini, I; Couture, M M; Donaire, A; Eltis, L D; Felli, I C; Luchinat, C; Piccioli, M; Rosato, A



High-throughput quantitation of amino acids in rat and mouse biological matrices using stable isotope labeling and UPLC-MS/MS analysis.  


Quantifying amino acids in biological matrices is typically performed using liquid chromatography (LC) coupled with fluorescent detection (FLD), requiring both derivatization and complete baseline separation of all amino acids. Due to its high specificity and sensitivity, the use of UPLC-MS/MS eliminates the derivatization step and allows for overlapping amino acid retention times thereby shortening the analysis time. Furthermore, combining UPLC-MS/MS with stable isotope labeling (e.g., isobaric tag for relative and absolute quantitation, i.e., iTRAQ) of amino acids enables quantitation while maintaining sensitivity, selectivity and speed of analysis. In this study, we report combining UPLC-MS/MS analysis with iTRAQ labeling of amino acids resulting in the elution and quantitation of 44 amino acids within 5 min demonstrating the speed and convenience of this assay over established approaches. This chromatographic analysis time represented a 5-fold improvement over the conventional HPLC-MS/MS method developed in our laboratory. In addition, the UPLC-MS/MS method demonstrated improvements in both specificity and sensitivity without loss of precision. In comparing UPLC-MS/MS and HPLC-MS/MS results of 32 detected amino acids, only 2 amino acids exhibited imprecision (RSD) >15% using UPLC-MS/MS, while 9 amino acids exhibited RSD >15% using HPLC-MS/MS. Evaluating intra- and inter-assay precision over 3 days, the quantitation range for 32 detected amino acids in rat plasma was 0.90-497 ?M, with overall mean intra-day precision of less than 15% and mean inter-day precision of 12%. This UPLC-MS/MS assay was successfully implemented for the quantitative analysis of amino acids in rat and mouse plasma, along with mouse urine and tissue samples, resulting in the following concentration ranges: 0.98-431 ?M in mouse plasma for 32 detected amino acids; 0.62-443 ?M in rat plasma for 32 detected amino acids; 0.44-8590?M in mouse liver for 33 detected amino acids; 0.61-1241 ?M in mouse kidney for 37 detected amino acids; and 1.39-1,681 ?M in rat urine for 34 detected amino acids. The utility of the assay was further demonstrated by measuring and comparing plasma amino acid levels between pre-diabetic Zucker diabetic fatty rats (ZDF/Gmi fa/fa) and their lean littermates (ZDF/Gmi fa/?). Significant differences (P<0.001) in 9 amino acid concentrations were observed, with the majority ranging from a 2- to 5-fold increase in pre-diabetic ZDF rats on comparison with ZDF lean rats, consistent with previous literature reports. PMID:24842860

Takach, Edward; O'Shea, Thomas; Liu, Hanlan



Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI  

PubMed Central

Background The anaerobe Clostridium difficile produces two major virulence factors toxin A and B that inactivate Rho proteins by glucosylation of a pivotal threonine residue. Purified toxins induce reorganization of the cytoskeleton and cell death in colonic cells. Whether all toxin effects on target cells depend on catalytic glucosyltransferase activity is unclear at present. Thus, we conducted a proteome approach to compare the protein profile of target cells treated either with wild type toxin A (rTcdA wt) or with a catalytically inactive mutant toxin A (mutant rTcdA). Relative protein quantification was feasible using isotope-coded protein labeling techniques (ICPL) and mass spectrometry (LC-MALDI). Results Altogether we found a significant differential expression of thirty proteins after treatment with rTcdA wt or mutant rTcdA. Mutant rTcdA caused up-regulation of seven proteins and sixteen proteins were responsive to rTcdA wt after 5 h. Long-term effect of rTcdA wt on protein expression was the down-regulation of eleven proteins. Up- or down-regulation of several proteins was verified by western blot analysis confirming the MS results. Conclusion Our results indicate incubation time-dependent effects of the clostridial glucosylating toxin A on colonic cells. The rTcdA wt impact more cellular functions than actin cytoskeleton reorganization and apoptosis. Furthermore, these data give insight into glucosyltransferase independent effects of clostridial glucosylating toxins on target cells after short incubation time. Additionally, our data reveal pro-inflammatory and proliferative effects of mutant rTcdA after short-term incubation. PMID:21849038



The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2  

PubMed Central

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with 13C3-D5-glycerol or 13C18-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that 13C3-D5-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, 13C18-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D5-glycerol to mice and measured plasma levels of D5-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D5-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered 13C18-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol. PMID:22493088

Qi, Jenson; Lang, Wensheng; Geisler, John G.; Wang, Ping; Petrounia, Ioanna; Mai, Selyna; Smith, Charles; Askari, Hossein; Struble, Geoffrey T.; Williams, Robyn; Bhanot, Sanjay; Monia, Brett P.; Bayoumy, Shariff; Grant, Eugene; Caldwell, Gary W.; Todd, Matthew J.; Liang, Yin; Gaul, Micheal D.; Demarest, Keith T.; Connelly, Margery A.



Determination of an Angiotensin II-regulated Proteome in Primary Human Kidney Cells by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)  

PubMed Central

Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models. PMID:23846697

Konvalinka, Ana; Zhou, Joyce; Dimitromanolakis, Apostolos; Drabovich, Andrei P.; Fang, Fei; Gurley, Susan; Coffman, Thomas; John, Rohan; Zhang, Shao-Ling; Diamandis, Eleftherios P.; Scholey, James W.



Transport of Indole-3-Butyric Acid and Indole-3-Acetic Acid in Arabidopsis Hypocotyls Using Stable Isotope Labeling1[C][W][OA  

PubMed Central

The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling. PMID:22323783

Liu, Xing; Barkawi, Lana; Gardner, Gary; Cohen, Jerry D.



Cytotoxicity, cytocompatibility, cell-labeling efficiency, and in vitro cellular magnetic resonance imaging of gadolinium-catalyzed single-walled carbon nanotubes  

PubMed Central

Cell tracking by magnetic resonance imaging (MRI) is an emerging technique that typically requires the use of MRI contrast agents (CAs). A MRI CA for cellular imaging should label cells efficiently at potentially safe concentrations, have high relaxivity, and not affect the cellular machinery. In this article, we report the cytotoxicity, cytocompatibility, and cell labeling efficiency in NIH/3T3 fibroblasts of novel, single-walled carbon nanotubes synthesized using gadolinium nano-particles as catalysts (Gd-SWCNTs). Cells incubated with the Gd-SWCNT showed a dose- (50–100 ?g/mL nanotube concentration) and time- (12–48 h) dependent decrease in viability. 30% cell death was observed for cells incubated with Gd-SWCNTs at the maximum dose of 100 ?g/mL for 48 h. Cells incubated with the Gd-SWCNTs at concentrations between 1–10 ?g/mL for 48 h showed no change in viability or proliferation compared to untreated controls. Additionally, at these potentially safe concentrations, up to 48 h, the cells showed no phosphatidyl serine externalization (pre-apoptotic condition), caspase-3 activity (point of no return for apoptosis), genetic damage, or changes in their division cycle. Localization of Gd-SWCNTs within the cells was confirmed by transmission electron microscopy (TEM) and Raman microscopy, and these results show 100% cell labeling efficiency. Elemental analysis also indicates significant uptake of Gd-SWCNTs by the cells (108–109 Gd3+ ions per cell). Finally, T1-weighted MRI at 3 T of Gd-SWCNT-labelled cells show up to a four-fold increase in MR signal intensities as compared to untreated cells. These results indicate that Gd-SWCNTs label cells efficiently at potentially safe concentrations, and enhance MRI contrast without any structural damage to the cells. PMID:23686792

Avti, Pramod K.; Caparelli, Elisabeth D.; Sitharaman, Balaji



Isotope ratio analysis of actinides, fission products, and geolocators by high-efficiency multi-collector thermal ionization mass spectrometry  

NASA Astrophysics Data System (ADS)

A ThermoFisher "Triton" multi-collector thermal ionization mass spectrometer (MC-TIMS) was evaluated for trace and ultra-trace level isotope ratio analysis of actinides (uranium, plutonium, and americium), fission products and geolocators (strontium, cesium, and neodymium). Total efficiencies (atoms loaded to ions detected) of up to 0.5-2% for U, Pu, and Am, and 1-30% for Sr, Cs, and Nd can be reported employing resin bead load techniques onto flat ribbon Re filaments or resin beads loaded into a millimeter-sized cavity drilled into a Re rod. This results in detection limits of <0.1 fg (104 atoms to 105 atoms) for 239-242+244Pu, 233+236U, 241-243Am, 89,90Sr, and 134,135,137Cs, and <=1 pg for natural Nd isotopes (limited by the chemical processing blank) using a secondary electron multiplier (SEM) or multiple-ion counters (MICs). Relative standard deviations (RSD) as small as 0.1% and abundance sensitivities of 1 × 106 or better using a SEM are reported here. Precisions of RSD [approximate]0.01-0.001% using a multi-collector Faraday cup array can be achieved at sub-nanogram concentrations for strontium and neodymium and are suitable to gain crucial geolocation information. The analytical protocols reported herein are of particular value for nuclear forensic and nuclear safeguard applications.

Bürger, S.; Riciputi, L. R.; Bostick, D. A.; Turgeon, S.; McBay, E. H.; Lavelle, M.



Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor.  


Although it is reported that metal and metal oxide nanoparticles, which are among the most rapidly commercialized materials, can cause toxicity to organisms, their fate in the environment and toxicity to marine organisms are not well understood. In this study, we used a stable isotope labelling approach to trace the fate of nanoparticles (NPs) in sediments and also investigated bio-uptake in two estuarine intra-sedimentary invertebrates Scrobicularia plana and Nereis diversicolor. We selected exposure to 3 mg kg(-1) sediment ZnO NPs since this level is a realistic prediction of the environmental concentration in sediments. 67ZnO NPs (DLS: 21-34 nm, positively charged: 31.3 mV) suspensions were synthesised in diethylene glycol (DEG). We explored the fate of 67ZnO NPs in sediment, 67Zn bioaccumulation and the biochemical (biomarkers of defence and damage) and behavioural (burrowing kinetics and feeding rates) biomarkers in both species to 67ZnO NPs and DEG on its own during a 16 d laboratory exposure. After exposure, 67Zn concentrations in sediment showed higher levels in the upper section (1cm: 2.59 mg kg(-1)) decreasing progressively (2 cm: 1.63 mg kg(-1), 3 cm: 0.90 mg kg(-1), 4 cm: 0.67 mg kg(-1)) to a minimum value at the bottom (5 cm: 0.31 mg kg(-1)). 67Zn bioaccumulation was observed in both organisms exposed to 67ZnO NPs in DEG but no major inter-species differences were found. At the biochemical level, 67ZnO NPs exposure significantly induced increased glutathione-S-transferase activity in worms and catalase activity in clams whereas superoxide dismutase activity and thiobarbituric acid reactive substance levels were not affected in any species. Exposure to DEG on its own leads to a significant increase of metallothionein-like protein levels in clams compared with those exposed to 67ZnO NPs or controls. Burrowing behaviour as well as feeding rate were significantly impaired in both species exposed to 67ZnO NPs. Concerning exposure to DEG on its own, burrowing behaviour impairments were also shown in both species and feeding rate was impaired in bivalves. At environmentally realistic concentration of 67ZnO NPs in sediment, there is no strong evidence for a severe nanoparticle effect since most effects were also observed in the presence of DEG alone. PMID:22858103

Buffet, Pierre-Emmanuel; Amiard-Triquet, Claude; Dybowska, Agnieszka; Risso-de Faverney, Christine; Guibbolini, Marielle; Valsami-Jones, Eugénia; Mouneyrac, Catherine



Absolute quantification of UGT1A1 in various tissues and cell lines using isotope label-free UPLC-MS/MS method determines its turnover number and correlates with its glucuronidation activities.  


Uridine 5'-diphosphate-glucuronosyltransferase (UGT)1A1 is a major phase II metabolism enzyme responsible for glucuronidation of drugs and endogenous compounds. The purpose of this study was to determine the expression level of UGT1A1 in human liver microsomes and human cell lines by using an isotope label-free LC-MS/MS method. A Waters Ultra performance liquid chromatography (UPLC) system coupled with an API 5500Qtrap mass spectrometer was used for the analysis. Two signature peptides (Pep-1, and Pep-2) were employed to quantify UGT1A1 by multiple reaction monitoring (MRM) approach. Standard addition method was used to validate the assay to account for the matrix effect. 17?-Estradiol was used as the marker substrate to determine UGT1A1 activities. The validated method has a linear range of 200-0.0195nM for both signature peptides. The precision, accuracy, and matrix effect were in acceptable ranges. UGT1A1 expression levels were then determined using 8 individual human liver microsomes, a pooled human liver microsomes, three UGT1A1 genotyped human liver microsomes, and four cell lines (Caco-2, MCF-7, Hela, and HepG2). The correlations study showed that the UGT1A1 protein levels were strongly correlated with its glucuronidation activities in human liver microsomes (R(2)=0.85) and in microsomes prepared from cell lines (R(2)=0.95). Isotope-labeled peptides were not necessary for LC-MS/MS quantitation of proteins. The isotope label-free absolute quantification method used here had good accuracy, sensitivity, linear range, and reproducibility, and were used successfully for the accurate determination of UGT1A1 from tissues and cell lines. PMID:24055854

Xu, Beibei; Gao, Song; Wu, Baojian; Yin, Taijun; Hu, Ming



Isotope-based analysis of modified tRNA nucleosides correlates modification density with translational efficiency.  


Useful diversity: Quantification of modified tRNA nucleobases in different murine and porcine tissues reveals a tissue-specific overall modification content. The modification content correlates with rates of protein synthesis in?vitro, suggesting a direct link between tRNA modification levels and tissue-specific translational efficiency. PMID:23037940

Brandmayr, Caterina; Wagner, Mirko; Brückl, Tobias; Globisch, Daniel; Pearson, David; Kneuttinger, Andrea Christa; Reiter, Veronika; Hienzsch, Antje; Koch, Susanne; Thoma, Ines; Thumbs, Peter; Michalakis, Stylianos; Müller, Markus; Biel, Martin; Carell, Thomas



Estimating Water Use Efficiency at the Watershed Scale Using Stable Isotopes  

Microsoft Academic Search

Ecosystem water use efficiency (WUE) is an important indicator of ecosystem processes, especially under drought conditions. Nocturnal cold air drainage provides an opportunity to monitor ecosystem WUE because as air flows downhill through a watershed, it collects respired CO2 from the soil and vegetation. Thus, sampling the CO2 concentration and delta13C throughout the cold air profile at the base of

K. Kavanagh; S. W. Blecker; J. D. Marshall



Carbon isotope assessment of intrinsic water use efficiency of C[sub 3] plants in two FACE experiments  

SciTech Connect

FACE (Free-Air CO[sub 2] Enrichment) provides a means for evaluating the direct effects of elevated Co[sub 2] on terrestrial ecosystems under field conditions. Intrinsic water use efficiency (IWUE), defined as A/g where A/g=C[sub a] (1-CC[sub i]/C[sub a])/1.6 with A being photosynthesis, g being conductance to water vapor and C[sub a] and C[sub i] being the CO[sub 2] concentration in the air and leaf respectively, was determined from the carbon isotope signatures of cotton and wheat, C[sub 3] plants, growing at ambient and elevated CO[sub 2] (550 ppm) in the field in Arizona. Sorghum and corn, C[sub 4] plants, served as monitors of [sup 13]C/[sup 12]C ratios of CO[sub 2] in air. Cotton and wheat exhibited increases in IWUE of 50% and 31% respectively, with a CO[sub 2] enrichment of 52%. Higher C[sub a]'s rather than large changes in C[sub i]/C[sub a] accounted for most of the increase in IWUE. Productivity of terrestrial ecosystems should rise with CO[sub 2] in the future as a consequence of increased water use efficiency.

Johnson, H.B.; Kimball, B.; Leavitt, S. (Univ. of Arizona, Tucson, AZ (United States))



Silicon isotopes indicate enhanced carbon export efficiency in the North Atlantic during deglaciation  

NASA Astrophysics Data System (ADS)

Today’s Sargasso Sea is nutrient starved, except for episodic upwelling events caused by wind-driven winter mixing and eddies. Enhanced diatom opal burial in Sargasso Sea sediments indicates that silicic acid, a limiting nutrient today, may have been more available in subsurface waters during Heinrich Stadials, millennial-scale climate perturbations of the last glacial and deglaciation. Here we use the geochemistry of opal-forming organisms from different water depths to demonstrate changes in silicic acid supply and utilization during the most recent Heinrich Stadial. We suggest that during the early phase (17.5-18?ka), wind-driven upwelling replenished silicic acid to the subsurface, resulting in low Si utilization. By 17?ka, stratification reduced the surface silicic acid supply leading to increased Si utilization efficiency. This abrupt shift in Si cycling would have contributed to high regional carbon export efficiency during the recent Heinrich Stadial, despite being a period of increasing atmospheric CO2.

Hendry, Katharine R.; Robinson, Laura F.; McManus, Jerry F.; Hays, James D.



Water use efficiency and carbon isotope composition of plants in a cold desert environment  

Microsoft Academic Search

The effects of the availabilities of water and nitrogen on water use efficiency (WUE) of plants were investigated in a sagebrush steppe. The four species studied wereArtemisia tridentata (shrub),Ceratoides lanata (suffrutescent shrub),Elymus lanceolatus (rhizomatous grass), andElymus elymoides (tussock grass). Water and nitrogen levels were manipulated in a two-by-two factorial design resulting in four treatments: control (no additions), added water, added

Nancee L. Toft; Jay E. Anderson; Robert S. Nowak



Nutritional efficiency of alpha-ketoisocaproate relative to leucine, assessed isotopically  

SciTech Connect

The efficiency of alpha-ketoisocaproate as a dietary substitute for leucine was assessed in rats by two techniques: first, the minimal dose of alpha-ketoisocaproate required, as a supplement to a leucine-free diet, to achieve a growth rate as great as animals receiving leucine was found to be between 2.2 and 4.4 times larger. Therefore the nutritional efficiency of alpha-ketoisocaproate lies between 0.23 and 0.46. Second, alpha-(1- UC)-ketoisocaproate and (TH)leucine were administered orally and the ratio of UC/TH incorporated into the leucine of whole-body protein and fibrin was measured. This ratio, divided by the ratio UC/TH injected, was the same in fibrin as in whole-body protein and averaged 0.39. Thus both techniques yield the same value, within the error of measurement, for the relative nutritional efficiency of alpha-ketoisocaproate. The authors also found that alpha-ketoisocaproate feeding at varying dosage did not alter this ratio in whole-body protein, suggesting that neither wide variations in growth rate nor exposure for 10 days to alpha-ketoisocaproate alters the relative rates of utilization (or oxidation) of alpha-ketoisocaproate vs. leucine.

Kang, C.W.; Walser, M.



Fluorescence energy transfer efficiency in labeled yeast cytochrome c: a rapid screen for ion biocompatibility in aqueous ionic liquids  

SciTech Connect

A fluorescence energy transfer de-quenching assay was implemented to follow the equilibrium unfolding behaviour of site-specific tetramethylrhodamine-labelled yeast cytochrome c in aqueous ionic liquid solutions; additionally, this approach offers the prospect of naked eye screening for biocompatible ion combinations in hydrated ionic liquids.

Baker, Sheila N [ORNL; Zhao, Hua [Savannah State University; Pandey, Siddharth [Indian Institute of Technology, Delhi; Heller, William T [ORNL; Bright, Frank [University of Buffalo, The State University of New York; Baker, Gary A [ORNL



Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis  

PubMed Central

The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO?), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z = 181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards. PMID:24736779

Seeley, Kent W.; Fertig, Alison R.; Dufresne, Craig P.; Pinho, Joao P. C.; Stevens, Stanley M.



Synthesis of (13) C2 (15) N2 -labeled anti-inflammatory and cytoprotective tricyclic bis(cyanoenone) ([(13) C2 (15) N2 ]-TBE-31) as an internal standard for quantification by stable isotope dilution LC-MS method.  


Tricyclic bis(cyanoenone), TBE-31, one of the most potent activators of the Keap1/Nrf2/antioxidant response element pathway, has been developed as a new anti-inflammatory and cytoprotective agent. (13) C2 (15) N2 -labeled TBE-31 ([(13) C2 (15) N2 ]-TBE-31), which has two (13) C and two (15) N atoms in two cyano groups, was designed to develop a method for quantification of cell, tissue, and plasma levels of TBE-31 that involves chromatography/mass spectrometry coupled with the use of a stable isotope-labeled internal standard. [(13) C2 (15) N2 ]-TBE-31 was successfully synthesized in four steps from a previously reported intermediate, which is prepared in 11 steps from cyclohexanone, by introduction of two (13) C atoms with ethyl [(13) C]formate and two (15) N atoms with hydroxyl[(15) N]amine. The stable isotope dilution liquid chromatography-mass spectrometry method for quantification of TBE-31 was successfully developed using [(13) C2 (15) N2 ]-TBE-31 to compensate for any variables encountered during sample processing and analysis. PMID:25196444

Zheng, Suqing; Huang, Jeffrey T-J; Knatko, Elena V; Sharp, Sheila; Higgins, Maureen; Ojima, Iwao; Dinkova-Kostova, Albena T; Honda, Tadashi



Carbon transfer from photosynthesis to below ground fine root/hyphae respiration in Quercus serrata using stable carbon isotope pulse labeling  

NASA Astrophysics Data System (ADS)

Studying carbon allocation in trees is a key to better understand belowground carbon cycle and its response to climate change. Tracing 13C in tree and soil compartments after pulse labeling is one of powerful tool to study the fate of carbon in forest ecosystems. This experiment was conducted in Yamashiro experimental forest, Kyoto, Japan. Annual mean temperature and precipitation from 1994 to 2009 are 15.5 ° C and 1,388 mm respectively. The branch pulse labeling were done 7 times in 2011 using same branch of Quercus serrata (H:11.7 m, DBH; 33.7 cm) to see seasonal variations of carbon velocity. Whole crown labeling of Quercus serrata (H:9 m, DBH; 13.7 cm) was done in 2012 to study carbon allocation and to especially focus on belowground carbon flux until to the hyphae respiration. Pure 13CO2 (99.9%) was injected to the labeling chamber which was set to branch or crown. Then, after one hour of branch labeling and 3.5 hour for crown labeling, the chamber was opened. Trunk respiration chambers, fine root chambers and hyphae chambers were set to the target tree to trace labeled carbon in the CO2 efflux. 41 ?m mesh was used to exclude ingrowth of roots into hyphae chambers. The results show that the velocity of carbon through the tree varied seasonally, with higher velocity in summer than autumn, averaging 0.47 m h-1. Half-lives of labeled carbon in autotrophic respiration were similar above and below ground during the growing season, but they were twice longer in trunk than in root in autumn. From the whole crown labeling done end of growing season, the 13CO2 signal was observed 25 hours after labeling in trunk chamber and 34-37.7 hours after labeling in fine root and hyphae respiration almost simultaneously. Half-lives of 13 was longer in trunk than below ground. Trunk respiration was still using labelled carbon during winter suggesting that winter trunk respiration is partly fueled by carbon stored in the trunk at the end of the growing season.

Dannoura, M.; Kominami, Y.; Takanashi, S.; Takahashi, K.



The Analysis on Influence of Main Factors on Theoretical Value of Energy Saving Rate for Energy Efficiency Labeling of Civil Buildings  

NASA Astrophysics Data System (ADS)

For typical residential buildings, no-large-scale and large-scale public buildings, according to China's Technical Guide for the Energy Efficiency Labeling of Civil Buildings, makes up missing data of the calculation benchmark and determines the boundary conditions for calculating the theoretical values of civil building energy efficiency. Based on equivalent full load hours method, develops a modular program and calculates building energy consumption for the demands of dynamic cooling and heating and lighting etc., finds out the corresponding relationship between star level's theoretical value of energy saving rate and specified-term limiting value in the Guide. With orthogonal experimental design and multiple linear regression, establishes the quantitative function of both the theoretical value of energy saving rate and main factors parameters, analyzes the impact of the control parameter on energy saving rate, and reveals the law of theoretical value of energy saving rate variation with the control parameter. For building energy efficiency labeling upgrade, presents technical measure need to be taken and analyses its feasibility. The results from the study can provide theoretical guidance for energy-saving design or retrofitting of civil buildings.

Wang, Zhiwei; Wang, Zhenling; Jiang, Bo; Zhang, Fan; Li, Peng; Cao, Wei


Transpiration efficiency over an annual cycle, leaf gas exchange and wood carbon isotope ratio of three tropical tree species.  


Variation in transpiration efficiency (TE) and its relationship with the stable carbon isotope ratio of wood was investigated in the saplings of three tropical tree species. Five individuals each of Platymiscium pinnatum (Jacq.) Dugand, Swietenia macrophylla King and Tectona grandis Linn. f. were grown individually in large (760 l) pots over 16 months in the Republic of Panama. Cumulative transpiration was determined by repeatedly weighing the pots with a pallet truck scale. Dry matter production was determined by destructive harvest. The TE, expressed as experiment-long dry matter production divided by cumulative water use, averaged 4.1, 4.3 and 2.9 g dry matter kg(-1) water for P. pinnatum, S. macrophylla and T. grandis, respectively. The TE of T. grandis was significantly lower than that of the other two species. Instantaneous measurements of the ratio of intercellular to ambient CO(2) partial pressures (c(i)/c(a)), taken near the end of the experiment, explained 66% of variation in TE. Stomatal conductance was lower in S. macrophylla than in T. grandis, whereas P. pinnatum had similar stomatal conductance to T. grandis, but with a higher photosynthetic rate. Thus, c(i)/c(a) and TE appeared to vary in response to both stomatal conductance and photosynthetic capacity. Stem-wood delta(13)C varied over a relatively narrow range of just 2.2 per thousand, but still explained 28% of variation in TE. The results suggest that leaf-level processes largely determined variation among the three tropical tree species in whole-plant water-use efficiency integrated over a full annual cycle. PMID:19661136

Cernusak, Lucas A; Winter, Klaus; Aranda, Jorge; Virgo, Aurelio; Garcia, Milton



Stable Carbon Isotope Composition (deltaC), Water Use Efficiency, and Biomass Productivity of Lycopersicon esculentum, Lycopersicon pennellii, and the F(1) Hybrid.  


Three tomatoes, Lycopersicon esculentum Mill. cv UC82B, a droughttolerant wild related species, Lycopersicon pennellii (Cor.) D'Arcy, and their F(1) hybrid, were grown in containers maintained at three levels of soil moisture. Season-long water use was obtained by summing over the season daily weight losses of each container corrected for soil evaporation. Plant biomass was determined by harvesting and weighing entire dried plants. Season-long water use efficiency (gram dry weight/kilogram H(2)O) was calculated by dividing the dry biomass by the season-long water use. The season-long water use efficiency was greatest in the wild parent, poorest in the domestic parent, and intermediate (but closer to the wild parent) in the F(1) hybrid. Instantaneous water-use efficiency (micromole CO(2)/millimole H(2)O) determined by gas exchange measurements on individual leaves was poorly correlated with season-long water use efficiency. However, the relative abundance of stable carbon isotopes of leaf tissue samples was strongly correlated with the season-long water use efficiency. Also, the isotopic composition and the season-long water use efficiency of each genotype alone were strongly negatively correlated with plant dry weight when the dry weight varied as a function of soil moisture. PMID:16666269

Martin, B; Thorstenson, Y R



Method development for the redox speciation analysis of iron by ion chromatography-inductively coupled plasma mass spectrometry and carryover assessment using isotopically labeled analyte analogues.  


An ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS) method was developed for the redox speciation analysis of iron (Fe) based on in-column complexation of Fe(2+) and Fe(3+) by dipicolinic acid (DPA). The effects of column type, mobile phase composition and molecular ion interference were studied in the method optimization. The carryover of the target species in the IC-ICP-MS method was uniquely and effectively evaluated using isotopically enriched analogues of the analytes ((54)Fe(2+) and (57)Fe(3+)). Standard solutions of the enriched standards were injected into the system following analysis of a sample, and the ratios of the isotopes of iron in the enriched standards were calculated based on the chromatographic peak areas. The concentrations of the analytes carried over from the sample to the enriched standards were determined using the quantitative relationship in isotope dilution mass spectrometry (IDMS). In contrast to the routine way of evaluating carryover effect by injecting a blank solution after sample analysis, the use of isotopically enriched standards identified significant analyte carryover in the present method. Extensive experiments were carried out to systematically identify the source of the carryover and to eliminate the problem; the separation column was found to be the exclusive source. More than 95% of the analyte carryover was eliminated by reducing the length of the column. The detection limit of the IC-ICP-MS method (MDL) for the iron species was 2ngg(-1). The method was used to determine Fe(2+) and Fe(3+) in synthetic aqueous standard solutions and a beverage sample. PMID:24819017

Wolle, Mesay Mulugeta; Fahrenholz, Timothy; Rahman, G M Mizanur; Pamuku, Matt; Kingston, H M 'Skip'; Browne, Damien



The application of high-resolution IR spectroscopy and isotope labeling for detailed investigation of TiO2/gas interface reactions  

NASA Astrophysics Data System (ADS)

The main motivation for the synthesis of Ti18O2 was the investigation of surface effects during titania/gas interaction. The interaction with carbon dioxide was investigated with the aim to explore oxygen isotope exchange at the Ti18,17O2/CO2 interface. For this purpose, high-resolution Fourier transform infrared spectroscopy of the gas phase was adopted. In the present study, we have explored the oxygen isotope exchange between gaseous C16O2 and solid Ti18O2 or Ti17O2. The present measurement in dark mixtures has, as its primary goal, the determination of the time-scale of the spontaneous isotope exchange between carbon dioxide and solid TiO2. The profiles of the individual lines of selected isotopologues (isolated lines in the spectrum) were fitted and quantified. The quantification of the spectra was carried out on the basis of calibration measurements of the absorption spectra of individual isotopologues (reference gases) of carbon dioxide at different pressures. The concentrations of individual isotopologues determined from the intensity profiles of the individual rotation-vibration lines are characterized by the exponential decrease of the 16O-C-16O isotopologue and the exponential increase of the 18O-C-18O isotopologue. The 18O-C-16O acts as an intermediate in the mixture and its concentration remains almost constant.

Civiš, Svatopluk; Ferus, Martin; Zukalová, Markéta; Kavan, Ladislav; Zelinger, Zden?k



Isotope effect in BEDT-TTF based organic superconductors  

SciTech Connect

The results of the comprehensive isotope effect studies, in which seven different isotopically labeled (involving {sup 13}C, {sup 34}S and {sup 2}H labeling) BEDT-TTF derivatives and isotopically labeled anion [Cu({sup 15}N{sup 13}CS){sub 2}]{sup {minus}} were utilized, are summarized. For the first time, convincing evidence for a genuine BCS-like mass isotope effect in an organic superconductor is revealed in these studies.

Kini, A.M.; Carlson, K.D.; Dudek, J.D.; Geiser, U.; Wang, H.H.; Williams, J.M.



Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin.  


The electronic structure and geometry of redox-active metal cofactors in proteins are tuned by the pattern of hydrogen bonding with the backbone peptide matrix. In this study we developed a method for selective amino acid labeling of a hyperthermophilic archaeal metalloprotein with engineered Escherichia coli auxotroph strains, and we applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance technique. Because deep electron spin-echo envelope modulation of two histidine (14)N(?) ligands of the cluster decreased non-coordinating (15)N signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which (14)N amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an (15)N-protein background. This has directly identified Lys45 N(?) as providing the major pathway for the transfer of unpaired electron spin density from the reduced cluster by a "through-bond" mechanism. All other backbone peptide nitrogens interact more weakly with the reduced cluster. The extension of this approach will allow visualizing the three-dimensional landscape of preferred pathways for the transfer of unpaired spin density from a paramagnetic metal center onto the protein frame, and will discriminate specific interactions by a "through-bond" mechanism from interactions which are "through-space" in various metalloproteins. PMID:23145461

Iwasaki, Toshio; Fukazawa, Risako; Miyajima-Nakano, Yoshiharu; Baldansuren, Amgalanbaatar; Matsushita, Shinichi; Lin, Myat T; Gennis, Robert B; Hasegawa, Kazuya; Kumasaka, Takashi; Dikanov, Sergei A



Seasonal variations in photosynthesis, intrinsic water-use efficiency and stable isotope composition of poplar leaves in a short-rotation plantation.  


Photosynthetic carbon assimilation and transpirational water loss play an important role in the yield and the carbon sequestration potential of bioenergy-devoted cultures of fast-growing trees. For six poplar (Populus) genotypes in a short-rotation plantation, we observed significant seasonal and genotypic variation in photosynthetic parameters, intrinsic water-use efficiency (WUEi) and leaf stable isotope composition (?13C and ?18O). The poplars maintained high photosynthetic rates (between 17.8 and 26.9??mol?m(-2)?s(-1) depending on genotypes) until late in the season, in line with their fast-growth habit. Seasonal fluctuations were mainly explained by variations in soil water availability and by stomatal limitation upon photosynthesis. Stomatal rather than biochemical limitation was confirmed by the constant intrinsic photosynthetic capacity (Vcmax) during the growing season, closely related to leaf nitrogen (N) content. Intrinsic water-use efficiency scaled negatively with carbon isotope discrimination (?13Cbl) and positively with the ratio between mesophyll diffusion conductance (gm) and stomatal conductance. The WUEi-?13Cbl relationship was partly influenced by gm. There was a trade-off between WUEi and photosynthetic N-use efficiency, but only when soil water availability was limiting. Our results suggest that seasonal fluctuations in relation to soil water availability should be accounted for in future modelling studies assessing the carbon sequestration potential and the water-use efficiency of woody energy crops. PMID:25074859

Broeckx, L S; Fichot, R; Verlinden, M S; Ceulemans, R



An Efficient and Compact Difference-Frequency-Generation Spectrometer and Its Application to 12CH3D/12CH4 Isotope Ratio Measurements  

PubMed Central

We have developed an efficient and compact 3.4 ?m difference-frequency-generation spectrometer using a 1.55 ?m distributed feedback (DFB) laser diode, a 1.06 ?m DFB laser diode, and a ridge-waveguide periodically poled lithium niobate. It is continuously tunable in the 30 cm?1 span and is applied to 12CH3D/12CH4 isotope ratio measurements. The suitable pair of 12CH3D ?4 pP(7,6) and 12CH4 ? 2+?4 R(6) F1(1) lines enabled us to determine their isotope ratio with a precision repeatability of 0.8‰ using a sample and a working standard of pure methane with an effective signal averaging time of 100 ms. PMID:22163569

Tsuji, Kiyoshi; Teshima, Hiroaki; Sasada, Hiroyuki; Yoshida, Naohiro



Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor  

PubMed Central

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (?1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 ?m width × 150 ?m depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation. PMID:18557614

Adams, Andre A.; Okagbare, Paul I.; Feng, Juan; Hupert, Matuesz L.; Patterson, Don; Gottert, Jost; McCarley, Robin L.; Nikitopoulos, Dimitris; Murphy, Michael C.; Soper, Steven A.



Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling1[W][OA  

PubMed Central

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state 13C-labeling experiments using [1-13C]-, [2-13C]-, and [U-13C6]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization. PMID:19939942

Masakapalli, Shyam K.; Le Lay, Pascaline; Huddleston, Joanna E.; Pollock, Naomi L.; Kruger, Nicholas J.; Ratcliffe, R. George



Pb and Sr isotope measurements by inductively coupled plasma mass spectrometer: efficient time management for precision improvement  

NASA Astrophysics Data System (ADS)

One of the factors limiting the precision of inductively coupled plasma mass spectrometry is the counting statistics, which depend upon acquisition time and ion fluxes. In the present study, the precision of the isotopic measurements of Pb and Sr is examined. The time of measurement is optimally shared for each isotope, using a mathematical simulation, to provide the lowest theoretical analytical error. Different algorithms of mass bias correction are also taken into account and evaluated in term of improvement of overall precision. Several experiments allow a comparison of real conditions with theory. The present method significantly improves the precision, regardless of the instrument used. However, this benefit is more important for equipment which originally yields a precision close to that predicted by counting statistics. Additionally, the procedure is flexible enough to be easily adapted to other problems, such as isotopic dilution.

Monna, F.; Loizeau, J.-L.; Thomas, B. A.; Guéguen, C.; Favarger, P.-Y.



Adrenal androgen 11. beta. - and 19-hydroxylation: a study on kinetic isotope effect and metabolic switching of /sup 3/H and /sup 14/C labeled substrates  

SciTech Connect

Monooxygenations at 11..beta.. and 19 are two main metabolic pathways of androstenedione (A) by sheep and dog adrenals and could be catalyzed by a single cytochrome P-450 as observed for deoxycorticosterone metabolism with the bovine adrenal cytochrome P-450/sub 11..beta../. They found an unusually high apparent kinetic isotope effect (k/sub H//k/sub T/ = 11.7) on the 19-hydroxylation of (19-/sup 3/H/sub 3/, 4-/sup 14/C)A with sheep and dog adrenal homogenates. They therefore attempted to assess the inverse secondary kinetic isotope effect of the simultaneously occurring 11..beta..-hydroxylation. The substrate ( and, /sup 3/H//sup 14/C = 15.1 and 21.9 for sheep and dog, respectively) was incubated with adrenal tissue homogenate in the presence of NADPH for 1-10 min. The products were mixed with carrier standards and purified through TLC, acetylation, and countercurrent distribution to show constant /sup 3/H//sup 14/C ratio. The /sup 3/H//sup 14/C ratios of the major (19-/sup 3/H/sub 3/, 4-/sup 14/C)-11..beta..-OHA were found to be elevated 1.1-1.2 fold whereas those of (19-/sup 3/H/sub 2/, 4-/sup 14/C)-19-OHA were decreased to 0.06 and the recovered A showed 0.95-0.99 of the initial ratio. While they observed a metabolic switching between the 2..beta..- and 19-hydroxylations of (19-/sup 3/H/sub 3/, 4-/sup 14/C)A with human placental aromatase (Fronckowiak and Osawa), it is suggested that adrenal non-aromatizing androgen 19-hydroxylation operates a metabolic switching with the 11..beta..-hydroxylation.

Osawa, Y.; Yarborough, C.



Hydrogen isotope fractionation during H2\\/CO2 acetogenesis: hydrogen utilization efficiency and the origin of lipid-bound hydrogen  

Microsoft Academic Search

Hydrogen metabolism was studied in the anaerobic bacterium, Sporomusa sp. strain DMG 58, by measuring natural abundance levels of deuterium in H 2 , H 2 O, and individual fatty acids during acetogenic growth on H 2 \\/ CO 2 . Four cultures were grown, each in medium with a distinct hydrogen-isotopic composition ( ? D-H 2 O). The ?




Biosynthesis of the iron-guanylylpyridinol cofactor of [Fe]-hydrogenase in methanogenic archaea as elucidated by stable-isotope labeling.  


[Fe]-hydrogenase catalyzes the reversible hydride transfer from H(2) to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H(2) and CO(2) in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe(II) is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme's cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of (13)C and (2)H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO(2). The metabolic origin of the two CO-ligands was CO(2) rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO(2) via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of (13)CO, the two CO-ligands and the acyl group became (13)C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized. PMID:22260087

Schick, Michael; Xie, Xiulan; Ataka, Kenichi; Kahnt, Jörg; Linne, Uwe; Shima, Seigo



Synthesis of deuterium-labeled prochlorperazine  

SciTech Connect

The propylpiperazine side chain of prochlorperazine was labeled with two, four, or six deuterium atoms by lithium aluminum deuteride reduction of the appropriate amide. The isotopic purity of the products after correcting for chlorine isotopes was greater than 95.7%.

Hawes, E.M.; Gurnsey, T.S.; Shetty, H.U.; Midha, K.K.



Assessment of effects of the rising atmospheric nitrogen deposition on nitrogen uptake and long-term water-use efficiency of plants using nitrogen and carbon stable isotopes.  


This study assesses the effects of the atmospheric nitrogen (N) deposition on the N uptake and the long-term water-use efficiency of two C(3) plants (Agropyron cristatum and Leymus chinensis) and two C(4) plants (Amaranthus retroflexus and Setaria viridis) using N and C stable isotopes. In addition, this study explores the potential correlation between leaf N isotope (?(15)N) values and leaf C isotope (?(13)C) values. This experiment shows that the atmospheric N deposition has significant effects on the N uptake, ?(15)N and leaf N content (N(m)) of C(3) plants. As the atmospheric N deposition rises, the proportion and the amount of N absorbed from the simulated atmospheric deposition become higher, and the ?(15)N and N(m) of the two C(3) plants both also increase, suggesting that the rising atmospheric N deposition is beneficial for C(3) plants. However, C(4) plants display different patterns in their N uptake and in their variations of ?(15)N and N(m) from those of C(3) plants. C(4) plants absorb less N from the atmospheric deposition, and the leaf N(m) does not change with the elevated atmospheric N deposition. Photosynthetic pathways may account for the differences between C(3) and C(4) plants. This study also shows that atmospheric N deposition does not play a role in determining the ?(13)C and in the long-term water-use efficiency of C(3) and C(4) plants, suggesting that the long-term water-use pattern of the plants does not change with the atmospheric N input. In addition, this study does not observe any relationship between leaf ?(15)N and leaf ?(13)C in both C(3) and C(4) plants. PMID:21638358

Yao, F Y; Wang, G A; Liu, X J; Song, L



18O Labeling over a Coffee Break: A Rapid Strategy for Quantitative Proteomics  

PubMed Central

Proteomics-based quantification methods for differential protein expression measurements are among the most important and challenging techniques in the field of mass spectrometry. Though numerous quantification methods have been established, no method meets all the demands for measuring accurate protein expression levels. Of the various relative quantification methods by isotopic labeling, 18O labeling method has been shown to be simple, specific, cost-effective and applicable to a wide range of analyses. However, some researchers refrain from using the method due to long incubation periods required during the labeling process. To address this problem, we demonstrate a method by which the labeling procedure can be completed in 15 min. We digested and labeled samples using immobilized trypsin on micro-spin columns to speed up the enzyme-mediated oxygen substitution, thereby completing the labeling process within 15 min with high labeling efficiency. We demonstrate the efficiency and accuracy of the method using a four protein mixture and whole cell lysate from rat vascular endothelial cells. PMID:18510357

Mirza, Shama P.; Greene, Andrew S.; Olivier, Michael



The use of isotopes in the determination of absolute bioavailability of drugs in humans.  


Absolute bioavailability studies in humans are not routinely performed as part of the drug registration process. They tend to be reasonably demanding, not least because toxicology data are required to support intravenous administration of a drug. Moreover, the classical crossover design of an absolute bioavailability study can suffer from artefacts caused by concentration-dependent pharmacokinetics. Many of the problems associated with absolute bioavailability studies can be alleviated using isotopically labelled drugs. Stable isotopes have been used in the performance of absolute bioavailability studies in humans for > 30 years. More recently, the advantages of using radiolabelled drugs have been expanded by using the ultrasensitive technology of accelerator mass spectrometry. Isotopic labelling not only allows for the accurate and efficient determination of absolute bioavailability, but can also provide information on first-pass effects and other pharmacokinetic parameters. PMID:16863443

Lappin, Graham; Rowland, Malcolm; Garner, R Colin



The Use of the Condensed Single Protein Production (cSPP) System for Isotope-Labeled Outer Membrane Proteins, OmpA and OmpX in E. coli  

PubMed Central

Gram-negative bacteria consist of two independent membranes, the inner cytoplasmic membrane and the outer membrane. The outer membrane contains a number of ?-barrel proteins such as OmpF, OmpC, OmpA and OmpX. In this paper, we explored to use the condensed Single Protein Production (cSPP) system for isotope labelling of OmpA and OmpX for NMR structural study, both of which are known to consist of eight ?-strands forming a barrel in the outer membrane. Using a deletion strain lacking all major outer membrane proteins, both OmpA and OmpX were expressed well in a 20-fold condensed SPP (cSPP) system. We demonstrated that outer membrane fractions prepared from the cSPP system in M9 medium containing 15-N-NH4Cl can be directly used for NMR structural study of the outer mebrane proteins without any further purification to get excellent [1H-15N]-TROSY spectra. PMID:20820947

Vaiphei, S. Thangminlal; Tang, Yuefeng; Montelione, Gaetano T.; Inouye, Masayori



Labeling Theory  

Microsoft Academic Search

Labeling theory provides a distinctively sociological approach that focuses on the role of social labeling in the development\\u000a of crime and deviance. The theory assumes that although deviant behavior can initially stem from various causes and conditions,\\u000a once individuals have been labeled or defined as deviants, they often face new problems that stem from the reactions of self\\u000a and others

Jón Gunnar Bernburg


Product Labels  

Microsoft Academic Search

In a self-service economy, a company must find factors to serve as surrogate salespeople to create attention for the product and distinguish it from competing items. Consumer desires for label information and how consumers are informed\\/misled by those statements were examined using wine labels. Eight hundred telephone interviews were completed across the United States.Consumers believed the information on wine labels

Dennis H. Tootelian; Karen Ross



Effect of drought on leaf gas exchange, carbon isotope discrimination, transpiration efficiency and productivity in field grown durum wheat genotypes .  

E-print Network

??Under drought prone conditions, wheat productivity is strongly related to photosynthetic activity and transpiration efficiency. In the present study, photosynthesis related traits were assessed at… (more)

Monneveux, Philippe



Nutrition label  

NSDL National Science Digital Library

This label shows that there are some nutrients that should be limited in the diet of humans and there are others that humans need to intake on a daily basis to stay healthy. These labels show the percent daily value that the food provides for each nutrient.

N/A N/A (FDA;)



Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.  


Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line. PMID:14714534

Qian, Wei-Jun; Goshe, Michael B; Camp, David G; Yu, Li-Rong; Tang, Keqi; Smith, Richard D



Tree-Ring Stable Isotopes Reveal Twentieth-Century Increases in Water-Use Efficiency of Fagus sylvatica and Nothofagus spp. in Italian and Chilean Mountains  

PubMed Central

Changes in intrinsic water use efficiency (iWUE) were investigated in Fagus sylvatica and Nothofagus spp. over the last century. We combined dendrochronological methods with dual-isotope analysis to investigate whether atmospheric changes enhanced iWUE of Fagus and Nothofagus and tree growth (basal area increment, BAI) along latitudinal gradients in Italy and Chile. Post-maturation phases of the trees presented different patterns in ?13C, ?13C, ?18O, Ci (internal CO2 concentration), iWUE, and BAI. A continuous enhancement in isotope-derived iWUE was observed throughout the twentieth century, which was common to all sites and related to changes in Ca (ambient CO2 concentration) and secondarily to increases in temperature. In contrast to other studies, we observed a general increasing trend of BAI, with the exception of F. sylvatica in Aspromonte. Both iWUE and BAI were uncoupled with the estimated drought index, which is in agreement with the absence of enduring decline in tree growth. In general, ?13C and ?18O showed a weak relationship, suggesting the major influence of photosynthetic rate on Ci and ?13C, and the minor contribution of the regulation of stomatal conductance to iWUE. The substantial warming observed during the twentieth century did not result in a clear pattern of increased drought stress along these latitudinal transects, because of the variability in temporal trends of precipitation and in specific responses of populations. PMID:25398040

Tognetti, Roberto; Lombardi, Fabio; Lasserre, Bruno; Cherubini, Paolo; Marchetti, Marco



Monte Carlo simulation of a PhosWatch detector using Geant4 for xenon isotope beta-gamma coincidence spectrum profile and detection efficiency calculations.  


A simulation tool has been developed using the Geant4 Toolkit to simulate a PhosWatch single channel beta-gamma coincidence detection system consisting of a CsI(Tl)/BC404 Phoswich well detector and pulse shape analysis algorithms implemented digital signal processor. The tool can be used to simulate the detector's response for all the gamma rays and beta particles emitted from (135)Xe, (133m)Xe, (133)Xe, (131m)Xe and (214)Pb. Two- and three-dimensional beta-gamma coincidence spectra from the PhosWatch detector can be produced using the simulation tool. The accurately simulated spectra could be used to calculate system coincidence detection efficiency for each xenon isotope, the corrections for the interference from the various spectral components from radon and xenon isotopes, and system gain calibration. Also, it can generate two- and three-dimensional xenon reference spectra to test beta-gamma coincidence spectral deconvolution analysis software. PMID:19647444

Mekarski, P; Zhang, W; Ungar, K; Bean, M; Korpach, E



Radioactively labelled porphyrin derivatives  

NASA Astrophysics Data System (ADS)

Radioactive labelling of guanidine-bearing tetraphenylporphyrin and Dy—texaphyrin with selected radionuclides (166Ho and 90Y) is described. A basic characterisation of studied porphyrin and texaphyrin, including their behaviour in a wide range of pH values and data on holmium and yttrium complexation with these compounds was probed using UV-VIS absorption spectrometry. The labelling yield of these macrocyclic molecules depends on the pH of the reaction mixture, metal: ligand ratio and time of incubation. Optimal reaction conditions for formation of porphyrin and texaphyrin radioactive complexes were determined by thin layer chromatography with the detection of ?- activity. The ability of porphyrin derivatives to bind anions was examined as well. Our experiments were focused on perrhenate ion (ReO4 -) because radiopharmaceuticals labelled with isotopes 186Re and 188Re play an important role in therapy of numerous tumour diseases. The possibility of applying ReO4 - anion directly for labelling purposes, without the necessity of its reduction to lower oxidation state, was not proved.

Koní?ová, R.; Ernestová, M.; Jedináková-K?ížová, V.; Král, V.



Rapid and efficient localization of depth electrodes and cortical labeling using free and open source medical software in epilepsy surgery candidates  

PubMed Central

Depth intracranial electrodes (IEs) placement is one of the most used procedures to identify the epileptogenic zone (EZ) in surgical treatment of drug resistant epilepsy patients, about 20–30% of this population. IEs localization is therefore a critical issue defining the EZ and its relation with eloquent functional areas. That information is then used to target the resective surgery and has great potential to affect outcome. We designed a methodological procedure intended to avoid the need for highly specialized medical resources and reduce time to identify the anatomical location of IEs, during the first instances of intracranial EEG recordings. This workflow is based on established open source software; 3D Slicer and Freesurfer that uses MRI and Post-implant CT fusion for the localization of IEs and its relation with automatic labeled surrounding cortex. To test this hypothesis we assessed the time elapsed between the surgical implantation process and the final anatomical localization of IEs by means of our proposed method compared against traditional visual analysis of raw post-implant imaging in two groups of patients. All IEs were identified in the first 24 H (6–24 H) of implantation using our method in 4 patients of the first group. For the control group; all IEs were identified by experts with an overall time range of 36 h to 3 days using traditional visual analysis. It included (7 patients), 3 patients implanted with IEs and the same 4 patients from the first group. Time to localization was restrained in this group by the specialized personnel and the image quality available. To validate our method; we trained two inexperienced operators to assess the position of IEs contacts on four patients (5 IEs) using the proposed method. We quantified the discrepancies between operators and we also assessed the efficiency of our method to define the EZ comparing the findings against the results of traditional analysis. PMID:24427112

Princich, Juan Pablo; Wassermann, Demian; Latini, Facundo; Oddo, Silvia; Blenkmann, Alejandro Omar; Seifer, Gustavo; Kochen, Silvia



Highly Efficient One-Pot Labeling of New Phosphonium Cations with Fluorine-18 as Potential PET Agents for Myocardial Perfusion Imaging.  


Lipophilic cations such as phosphonium salts can accumulate in mitochondria of heart in response to the negative inner-transmembrane potentials. Two phosphonium salts [(18)F]FMBTP and [(18)F]mFMBTP were prepared and evaluated as potential myocardial perfusion imaging (MPI) agents in this study. The cations were radiolabeled via a simplified one-pot method starting from [(18)F]fluoride and followed by physicochemical property tests, in vitro cellular uptake assay, ex vivo mouse biodistribution, and in vivo rat microPET imaging. The total radiosynthesis time was less than 60 min including HPLC purification. The [(18)F] labeled compounds were obtained in high radiolabeling yield (?50%) and good radiochemical purity (>99%). Both compounds were electropositive, and their log P values at pH 7.4 were 1.16 ± 0.003 (n = 3) and 1.05 ± 0.01 (n = 3), respectively. Both [(18)F]FMBTP and [(18)F]mFMBTP had high heart uptake (25.24 ± 2.97% ID/g and 31.02 ± 0.33% ID/g at 5 min postinjection (p.i.)) in mice with good retention (28.99 ± 3.54% ID/g and 26.82 ± 3.46% ID/g at 120 min p.i.). From the PET images in rats, the cations exhibited high myocardium uptake and fast clearance from liver and small intestine to give high-contrast images across all time points. These phosphonium cations were radiosynthesized via a highly efficient one-pot procedure for potential MPI offering high heart accumulation and rapid nontarget clearance. PMID:24852080

Zhao, Zuoquan; Yu, Qian; Mou, Tiantian; Liu, Chang; Yang, Wenjiang; Fang, Wei; Peng, Cheng; Lu, Jie; Liu, Yu; Zhang, Xianzhong



18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry  

SciTech Connect

Selected reaction monitoring-mass spectrometry (SRM-MS) is an emerging technology for high throughput targeted protein quantification and verification in biological and biomarker discovery studies; however, the cost associated with the use of stable isotope labeled synthetic peptides as internal standards is prohibitive for quantitatively screening large numbers of candidate proteins as often required in the pre-verification phase of biomarker discovery. Herein we present the proof-of-concept experiments of using an 18O-labeled 'universal' reference as comprehensive internal standards for quantitative SRM-MS analysis. With an 18O-labeled whole proteome sample as reference, every peptide of interest will have its own corresponding heavy isotope labeled internal standard, thus providing an ideal approach for quantitative screening of a large number of candidates using SRM-MS. Our results showed that the 18O incorporation efficiency using a recently improved protocol was >99.5% for most peptides investigated, a level comparable to 13C/15N labeled synthetic peptides in terms of heavy isotope incorporation. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into mouse plasma with an 18O-labeled mouse plasma reference. A dynamic range of four orders of magnitude in relative concentration was obtained with high reproducibility (i.e., coefficient of variance <10%) based on the 16O/18O peak area ratios. Absolute and relative quantification of C-reactive protein and prostate-specific antigen were demonstrated by coupling an 18O-labeled reference with standard additions of protein standards. Collectively, our results demonstrated that the use of 18O-labeled reference provides a convenient and effective strategy for quantitative SRM screening of large number of candidate proteins.

Kim, Jong Seo; Fillmore, Thomas L.; Liu, Tao; Robinson, Errol W.; Hossain, Mahmud; Champion, Boyd L.; Moore, Ronald J.; Camp, David G.; Smith, Richard D.; Qian, Weijun



Quantitative proteome analysis of cisplatin-induced apoptotic Jurkat T cells by stable isotope labeling with amino acids in cell culture, SDS-PAGE, and LC-MALDI-TOF/TOF MS.  


Quantitative proteome analysis of cisplatin-induced apoptosis in total Jurkat T cell lysates was performed in order to identify modified proteins. Proteins were labeled in cell culture with stable isotopes of arginines, and fractionated by SDS-PAGE. Subsequently, tryptic peptides were analyzed by nano-LC coupled offline to MALDI-TOF/TOF-MS as an alternative to commonly used online LC-ESI-MS. As a result, 26 proteins were found with a relative abundance higher than 1.5, thereof 19 already known and seven unknown to be involved in apoptosis (adenine phosphoribosyltransferase, microsomal signal peptidase 25 kDa subunit, phosphomevalonate kinase, probable rRNA processing protein EBP2, RNA-binding protein 4, transmembrane protein 33, and tetratricopeptide repeat domain 9C). Immunoblotting of core-binding factor beta and elongation factor 2 revealed similar quantitative changes as detected by the SILAC-based proteomics approach. Strikingly, 8 of 26 identified apoptosis-modified proteins contained at least one RNA-binding motif. Three caspase cleavage sites of the 54 kDa nuclear RNA-binding protein (p54nrb) were mapped at DQLD(231) (downward arrow)D, DQVD(286) (downward arrow)R, and MMPD(422) (downward arrow)G by applying caspase-3 to the in vitro translated protein and mutation analysis. The determined caspase cleavage sites were located C-terminal to the two RNA-binding motifs and one (DQLD(231) (downward arrow)D) within the NOPS domain of p54nrb. Concisely, quantitative protein data generated by offline LC-MALDI-MS were shown to be particularly accurate. Furthermore, only regulated peptides were selected in a result-dependent manner for MS/MS analyses and revealed novel apoptosis-modified proteins. PMID:17987630

Schmidt, Frank; Hustoft, Hanne K; Strozynski, Margarita; Dimmler, Christiane; Rudel, Thomas; Thiede, Bernd



Quantitative Proteomics Using Stable Isotope Labeling with Amino Acids in Cell Culture Reveals Changes in the Cytoplasmic, Nuclear, and Nucleolar Proteomes in Vero Cells Infected with the Coronavirus Infectious Bronchitis Virus*  

PubMed Central

Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis. Here we detail a high throughput quantitative proteomics analysis of Vero cells infected with the coronavirus infectious bronchitis virus (IBV), a positive strand RNA virus that replicates in the cytoplasm. Stable isotope labeling with amino acids in cell culture (SILAC) was used in conjunction with LC-MS/MS to identify and quantify 1830 cellular and two viral proteins from IBV-infected cells. Fractionation of cells into cytoplasmic, nuclear, and nucleolar extracts was used to reduce sample complexity and provide information on the trafficking of proteins between the different compartments. Each fraction showed a proportion of proteins exhibiting ?2-fold changes in abundance. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be grouped into different functional categories. These included proteins regulated by NF-?B- and AP-1-dependent pathways and proteins involved in the cytoskeleton and molecular motors. A luciferase-based reporter gene assay was used to validate the up-regulation of AP-1- and NF-?B-dependent transcription in IBV-infected cells and confirmed using immunofluorescence. Immunofluorescence was used to validate changes in the subcellular localization of vimentin and myosin VI in IBV-infected cells. The proteomics analysis also confirmed the presence of the viral nucleocapsid protein as localizing in the cytoplasm, nucleus, and nucleolus and the viral membrane protein in the cytoplasmic fraction. This research is the first application of SILAC to study total host cell proteome changes in response to positive sense RNA virus infection and illustrates the versatility of this technique as applied to infectious disease research. PMID:20467043

Emmott, Edward; Rodgers, Mark A.; Macdonald, Andrew; McCrory, Sarah; Ajuh, Paul; Hiscox, Julian A.



Total efficiency of an isotope-separator-on-line production system based on an electron cyclotron resonance ion source associated with a carbon target: The case of SPIRAL 1  

SciTech Connect

An original approach to the time behavior of an isotope-separator-on-line production system is proposed in the case of a production system where the target and the ion source are connected through a conductance much larger than that of the exit hole of the source. One major goal of this article is to derive the analytical expression of the response time of the system for noble gases from statistical parameters only, which can be deduced from a few simple measurements. The validity limits of the expression of the total efficiency are given, and the calculations are compared to the results obtained at GANIL during operation of SPIRAL 1, using a carbon target close coupled to a multicharged electron cyclotron resonance ion source. The final analytical expression for the total efficiency shows that the usual product of diffusion efficiency, effusion efficiency, and ionization efficiency cannot be applied in our case. We show how it is possible to predict the atom-to-ion transformation efficiency for radioactive isotopes of noble gas using response times measured for stable isotopes.

Jardin, P.; Eleon, C.; Farabolini, W.; Boilley, D.; Dubois, M.; Gaubert, G.; Cornell, J.C.; Huet-Equilbec, C.; Lecesne, N.; Leroy, R.; Pacquet, J.Y.; Saint Laurent, M.G.; Villari, A.C.C. [Grand Accelerateur National d' Ions Lourds (GANIL), Boulevard Henri Becquerel, B. P. 55027, 14076 Caen Cedex 5 (France); Centre d' Etudes Nucleaires (CEN) de Saclay, 91191 Gif sur Yvette Cedex (France); Grand Accelerateur National d' Ions Lourds (GANIL), Boulevard Henri Becquerel, B. P. 55027, 14076 Caen Cedex 5 (France)



Procedure and Device for the Separation of an Isotope by Selective Ionization.  

National Technical Information Service (NTIS)

A procedure for the separation of isotopes by selective ionization and which efficiently uses an isotope source is described. A promising technique for higher yield separation of the exp 235 U isotope includes selective ionization of this isotope in urani...

A. Kantrowitz



Multiple tag labeling method for DNA sequencing  


A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

Mathies, Richard A. (Contra Costa County, CA); Huang, Xiaohua C. (Mt. View, CA); Quesada, Mark A. (San Francisco, CA)



Correlated optical and isotopic nanoscopy  

PubMed Central

The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100?nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures. PMID:24718107

Saka, Sinem K.; Vogts, Angela; Krohnert, Katharina; Hillion, Francois; Rizzoli, Silvio O; Wessels, Johannes T.



A comprehensive human computation framework: with application to image labeling  

Microsoft Academic Search

Image and video labeling is important for computers to understand images and videos and for image and video search. Manual labeling is tedious and costly. Automatically image and video labeling is yet a dream. In this paper, we adopt a Web 2.0 approach to labeling images and videos efficiently: Internet users around the world are mobilized to apply their \\

Yang Yang; Bin B. Zhu; Rui Guo; Linjun Yang; Shipeng Li; Nenghai Yu



1. Isotope Definitions and terms a) Isotopes and isotope ratios.  

E-print Network

3/24/2011 1 Outline 1. Isotope Definitions and terms a) Isotopes and isotope ratios. Isotopes fractionation c) Simple illustration with the water cycle 2. CO2 isotopes in photosynthesis a) Photosynthetic discrimination in C3 plants b) C3 vs C4 photosynthesis and the distinction in isotopes c) Measuring isotopic

Saleska, Scott


Assessing the Cr(VI) reduction efficiency of a permeable reactive barrier using Cr isotope measurements and 2D reactive transport modeling  

NASA Astrophysics Data System (ADS)

In Thun, Switzerland, a permeable reactive barrier (PRB) for Cr(VI) reduction by gray cast iron was installed in May 2008. The PRB is composed of a double array of vertical piles containing iron shavings and gravel. The aquifer in Thun is almost saturated with dissolved oxygen and the groundwater flow velocities are ca. 10-15 m/day. Two years after PRB installation Cr(VI) concentrations still permanently exceed the Swiss threshold value for contaminated sites downstream of the barrier at selected localities. Groundwater ?53/52CrSRM979 measurements were used to track Cr(VI) reduction induced by the PRB. ?53/52CrSRM979 values of two samples downstream of the PRB showed a clear fractionation towards more positive values compared to four samples from the hotspot, which is clear evidence of Cr(VI) reduction induced by the PRB. Another downstream sample did not show a shift to more positive ?53/52CrSRM979 values. Because this latter location correlates with the highest downstream Cr(VI) concentration it is proposed that a part of the Cr(VI) plume is bypassing the barrier. Using a Rayleigh fractionation model a minimum present-day overall Cr(VI) reduction efficiency of ca. 15% was estimated. A series of 2D model simulations, including the fractionation of Cr isotopes, confirm that only a PRB bypass of parts of the Cr(VI) plume can lead to the observed values. Additionally, the simulations revealed that the proposed bypass occurs due to an insufficient permeability of the individual PRB piles. It is concluded that with this type of PRB a complete and long-lasting Cr(VI) reduction is extremely difficult to achieve for Cr(VI) contaminations located in nearly oxygen and calcium carbonate saturated aquifer in a regime of high groundwater velocities. Additional remediation action would limit the environmental impact and allow to reach target concentrations.

Wanner, Christoph; Zink, Sonja; Eggenberger, Urs; Mäder, Urs




EPA Science Inventory

The report gives results of an investigation of the use of stable isotopically labeled compounds as internal standards for quantitative isotope dilution GC/MS determinations. The availability of labeled compounds and the costs associated with using them for routine analyses were ...


Tritium removal by isotopic exchange  

NASA Astrophysics Data System (ADS)

This paper discusses how isotopic replacement is effective to remove tritium retained in the plasma facing surface in a DT reactor based on our recent studies on retention of hydrogen isotopes (H, D, and T) in plasma facing carbon tiles used in JT-60U. The isotope ratio of D and H in hydrogen retained near surface layers of the plasma facing wall is easily equilibrated with the flux ratio of hydrogen isotopes impinging the surface. Therefore DD discharges after DT discharges would effectively remove T retained in the surface layers during DT discharges. The efficiency of the replacement is higher and deeper for higher temperatures.

Tanabe, T.; Sugiyama, K.; Shibahara, T.; Hirohata, Y.; Yoshida, M.; Masaki, K.; Sato, M.



FRAM (Fixed Energy, Response Function Analysis with Multiple Efficiency): A new, versatile gamma-ray spectrometry code for measuring the isotopic composition of plutonium  

SciTech Connect

We describe the characteristics and features and demonstrate the performance of a new code (FRAM) for determining the isotopic composition of plutonium using gamma-ray spectroscopy. This versatile code can measure an extremely wide range of isotopic compositions and is extremely easy to tailor to specialized measurement conditions. Measurement precision, accuracy, and throughput are significantly improved over previous Los Alamos National Laboratory (LANL) codes. 13 refs., 2 figs., 4 tabs.

Sampson, T.E.; Nelson, G.W.; Kelley, T.A.



Photosynthetic carbon isotope discrimination and its relationship to the carbon isotope signals of stem, soil and ecosystem respiration (Invited)  

Microsoft Academic Search

Photosynthetic carbon (C) isotope discrimination labels photosynthates (deltaA) and atmospheric CO2 (deltaa) with variable C isotope compositions during fluctuating environmental conditions. In this context, the C isotope composition of respired CO2 within ecosystems is often hypothesized to vary temporally with photosynthetic discrimination. We investigated the relationship between photosynthetic discrimination and the C isotope signals from stem (deltaW), soil (deltaS) and

Lisa Wingate; Jérôme Ogée; Régis Burlett; Alexandre Bosc; Marion Devaux; John Grace; Denis Loustau; Arthur Gessler



Simultaneous tracing of {sup 76}Se-selenite and {sup 77}Se-selenomethionine by absolute labeling and speciation  

SciTech Connect

Nutritional selenocompounds are transformed into the assumed common intermediate selenide, which is utilized for the synthesis of selenoenzymes or transformed into methylated metabolites for excretion. Hence, selenocompound metabolites can be traced only with labeled selenium. Here we applied a new tracer method for the metallomics of biometals using simultaneous speciation of each metallome labeled with different homo-elemental isotopes to metabolism and availability of selenium. Rats were depleted of endogenous natural abundance selenium by feeding a single selenium stable isotope ({sup 82}Se-selenite) and then administered {sup 76}Se-selenite and {sup 77}Se-selenomethionine ({sup 77}Se-SeMet)simultaneously. Biological samples were subjected to quantification and speciation analysis by HPLC-ICPMS. Metabolites of the labeled {sup 76}Se and {sup 77}Se and interaction with endogenous selenium were traced and examined without interference from the corresponding endogenous natural abundance isotopes. Differences in the distribution and metabolism among organs and between the two nutritional selenocompounds were compared under exactly identical biological and analytical conditions: (1) selenite was distributed more efficiently than SeMet in organs and body fluids except the pancreas. (2) SeMet was taken up by organs in its intact form. (3) Selenium of SeMet origin was distributed selectively in the pancreas and mostly bound to a protein together with intact SeMet. (4) Selenosugars A and B but not trimethylselenonium (TMSe) were detected in the liver. (5) Selenosugar B and TMSe were detected in the kidneys.

Suzuki, Kazuo T. [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan)]. E-mail:; Somekawa, Layla [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan); Kurasaki, Kazuki [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan); Suzuki, Noriyuki [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan)



Simultaneous tracing of 76Se-selenite and 77Se-selenomethionine by absolute labeling and speciation.  


Nutritional selenocompounds are transformed into the assumed common intermediate selenide, which is utilized for the synthesis of selenoenzymes or transformed into methylated metabolites for excretion. Hence, selenocompound metabolites can be traced only with labeled selenium. Here we applied a new tracer method for the metallomics of biometals using simultaneous speciation of each metallome labeled with different homo-elemental isotopes to metabolism and availability of selenium. Rats were depleted of endogenous natural abundance selenium by feeding a single selenium stable isotope ((82)Se-selenite) and then administered (76)Se-selenite and (77)Se-selenomethionine ((77)Se-SeMet)simultaneously. Biological samples were subjected to quantification and speciation analysis by HPLC-ICPMS. Metabolites of the labeled (76)Se and (77)Se and interaction with endogenous selenium were traced and examined without interference from the corresponding endogenous natural abundance isotopes. Differences in the distribution and metabolism among organs and between the two nutritional selenocompounds were compared under exactly identical biological and analytical conditions: (1) selenite was distributed more efficiently than SeMet in organs and body fluids except the pancreas. (2) SeMet was taken up by organs in its intact form. (3) Selenium of SeMet origin was distributed selectively in the pancreas and mostly bound to a protein together with intact SeMet. (4) Selenosugars A and B but not trimethylselenonium (TMSe) were detected in the liver. (5) Selenosugar B and TMSe were detected in the kidneys. PMID:16956638

Suzuki, Kazuo T; Somekawa, Layla; Kurasaki, Kazuki; Suzuki, Noriyuki



Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics  

SciTech Connect

Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample. Peptide (18)O labeling is ideally suited for generating a labeled "universal" reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics.

Qian, Weijun; Petritis, Brianne O.; Nicora, Carrie D.; Smith, Richard D.



Aircraft profile measurements of 18O/16O and D/H isotope ratios of cloud condensate and water vapor constrain precipitation efficiency and entrainment rates in tropical clouds  

NASA Astrophysics Data System (ADS)

Convective clouds play a significant role in the moisture and heat balance of the tropics. The dynamics of organized and isolated convection are a function of the background thermodynamic profile and wind shear, buoyancy sources near the surface and the latent heating inside convective updrafts. The stable oxygen and hydrogen isotope ratios in water vapor and condensate can be used to identify dominant moisture exchanges and aspects of the cloud microphysics that are otherwise difficult to observe. Both the precipitation efficiency and the dilution of cloud updrafts by entrainment can be estimated since the isotopic composition outside the plume is distinct from inside. Measurements of the 18O/16O and D/H isotope ratios were made in July 2011 on 13 research flights of the NCAR C130 aircraft during the ICE-T (Ice in Clouds Experiment - Tropical) field campaign near St Croix. Measurements were made using an instrument based on the Picarro Wave-Length Scanning Cavity Ring Down platform that includes a number of optical, hardware and software modifications to allow measurements to be made at 5 Hz for deployment on aircraft. The measurement system was optimized to make precise measurements of the isotope ratio of liquid and ice cloud condensate by coupling the gas analyzer to the NCAR Counter flow Virtual Impactor inlet. The inlet system provides a particle enhancement while rejecting vapor. Sample air is vigorously heated before flowing into the gas phase analyzer. We present statistics that demonstrate the performance and calibration of the instrument. Measured profiles show that environmental air exhibits significant layering showing controls from boundary layer processes, large scale horizontal advection and regional subsidence. Condensate in clouds is consistent with generally low precipitation efficiency, although there is significant variability in the isotope ratios suggesting heterogeneity within plumes and the stochastic nature of detrainment processes. Entrainment of air into the plume is seen as evaporation of condensate. In the plume between about -7 and -12C, the ice condensate fraction increases with height, and the isotope ratios are used to discern ice formation from deposition from ice formed from in situ freezing of cloud liquid. The observed profiles demonstrate a new capacity for cloud process studies and provide new insight into the water budget of clouds.

Noone, D. C.; Raudzens Bailey, A.; Toohey, D. W.; Twohy, C. H.; Heymsfield, A.; Rella, C.; Van Pelt, A. D.



Comparison of various rhenium-188-labeled diphosphonates for the treatment of bone metastases  

Microsoft Academic Search

In the past, many diphosphonates were introduced as bone scan radiopharmaceuticals. In addition, diphosphonates have been labeled with beta-emitted isotopes and developed into useful therapeutic drugs for bone metastases. However, it is not clear which diphosphonate is the best choice when labeling with Re-188. In this study, we labeled methylene diphosphonate (MDP), hydroxyethylidene diphosphonate (HEDP), and hydroxymethane diphosphonate (HDP) with

Bor-Tsung Hsieh; Jih-Fang Hsieh; Shih-Chuan Tsai; Wan-Yu Lin; Shyh-Jen Wang; Gann Ting




Microsoft Academic Search

A process for separating isotopes of an element is outlined which is ; efficient and does not require large amounts of energy. The process comprises ; subjecting the mixture of a compound of the isotopes to monochromatic radiation ; at a wavelength corresponding to the molecular resonance peak of the compound of ; the desired isotope, and separating the dissociated




Long-term tree growth rate, water use efficiency, and tree ring nitrogen isotope composition of Pinus massoniana L. in response to global climate change and local nitrogen deposition in Southern China  

Microsoft Academic Search

Purpose  We aimed to investigate long-term tree growth rates, water use efficiencies (WUE), and tree ring nitrogen (N) isotope compositions\\u000a (?15N) of Masson pine (Pinus massoniana L.) in response to global climate change and local N deposition in Southern China.\\u000a \\u000a \\u000a \\u000a \\u000a Materials and methods  Tree annual growth rings of Masson pine were collected from four forest sites, viz. South China Botanical Garden (SBG),

Fangfang Sun; Yuanwen Kuang; Dazhi Wen; Zhihong Xu; Jianli Li; Weidong Zuo; Enqing Hou



Liquid chromatography combined with mass spectrometry for 13C isotopic analysis in life science research.  


Among the different disciplines covered by mass spectrometry, measurement of (13)C/(12)C isotopic ratio crosses a large section of disciplines from a tool revealing the origin of compounds to more recent approaches such as metabolomics and proteomics. Isotope ratio mass spectrometry (IRMS) and molecular mass spectrometry (MS) are the two most mature techniques for (13)C isotopic analysis of compounds, respectively, for high and low-isotopic precision. For the sample introduction, the coupling of gas chromatography (GC) to either IRMS or MS is state of the art technique for targeted isotopic analysis of volatile analytes. However, liquid chromatography (LC) also needs to be considered as a tool for the sample introduction into IRMS or MS for (13)C isotopic analyses of non-volatile analytes at natural abundance as well as for (13)C-labeled compounds. This review presents the past and the current processes used to perform (13)C isotopic analysis in combination with LC. It gives particular attention to the combination of LC with IRMS which started in the 1990's with the moving wire transport, then subsequently moved to the chemical reaction interface (CRI) and was made commercially available in 2004 with the wet chemical oxidation interface (LC-IRMS). The LC-IRMS method development is also discussed in this review, including the possible approaches for increasing selectivity and efficiency, for example, using a 100% aqueous mobile phase for the LC separation. In addition, applications for measuring (13)C isotopic enrichments using atmospheric pressure LC-MS instruments with a quadrupole, a time-of-flight, and an ion trap analyzer are also discussed as well as a LC-ICPMS using a prototype instrument with two quadrupoles. PMID:17853432

Godin, Jean-Philippe; Fay, Laurent-Bernard; Hopfgartner, Gérard



Colloidal palladium particles of different shapes for electron microscopy labeling.  


The immunogold technique is a valuable method for labeling cellular macromolecules. However, multiple labeling using colloidal gold (cAu) nanoparticles of different sizes presents certain drawbacks; namely, as particle size increases, there is a decreased labeling efficiency and diminished spatial resolution with respect to the locations of labeled epitopes. Both concerns also limit the utility of heavy metal particles for comparative analysis of labeling densities. To minimize the variables due to differential labeling efficiencies, the best solution would be to conduct multiple labeling with particles of similar size. Consequently, some parameter other than size is necessary to distinguish each label type. In this study, we report the synthesis of colloidal palladium (cPd) nanoparticles of similar size but having two distinct shapes, umbonate and faceted, which are readily distinguishable from spherical colloidal gold particles. Their utility and fidelity as labels using a human platelet whole-mount model is also demonstrated. PMID:20030909

Meyer, Daryl A; Oliver, Julie A; Albrecht, Ralph M



In vitro comparison of HMPAO and gentisic acid for labelling leukocytes with 99mTc.  


Leukocytes can be labelled with 99mTc using HMPAO and gentisic acid methods. We compared the two methods with respect to labelling efficiency on mixed leukocytes and isolated polymorphonuclear (PMN) and mononuclear (MN) cells, and the in vitro stability of the label. HMPAO produced approximately 70% labelling efficiency on mixed or PMN cells and the label was stable in saline or plasma. Labelling efficiency on MN was only 14% and was less stable. Gentisic acid produced a labelling efficiency of 52% on PMN and 35% on MN; both were stable in saline but less stable in plasma. In conclusion, HMPAO produces higher labelling efficiency and the label shows greater in vitro stability in plasma. However, gentisic acid is much less expensive to use, allows labelling of MN cells, and should result in more favourable microdosimetry. Preliminary clinical results suggest that gentisic acid is equivalent to HMPAO but has the advantage of being much cheaper. PMID:2161770

Ecclestone, M; Proulx, A; Ballinger, J R; Gerson, B; Reid, R H; Gulenchyn, K Y



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

ERIC Educational Resources Information Center

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

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



A fully enzymatic method for site-directed spin labeling of long RNA  

PubMed Central

Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5?-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies. PMID:24981512

Lebars, Isabelle; Vileno, Bertrand; Bourbigot, Sarah; Turek, Philippe; Wolff, Philippe; Kieffer, Bruno



Atom trap trace analysis of krypton isotopes  

SciTech Connect

A new method of ultrasensitive isotope trace analysis has been developed. This method, based on the technique of laser manipulation of neutral atoms, has been used to count individual {sup 85}Kr and {sup 81}Kr atoms present in a natural krypton gas sample with isotopic abundances in the range of 10{sup {minus}11} and 10{sup {minus}13}, respectively. This method is free of contamination from other isotopes and elements and can be applied to several different isotope tracers for a wide range of applications. The demonstrated detection efficiency is 1 x 10{sup {minus}7}. System improvements could increase the efficiency by many orders of magnitude.

Bailey, K.; Chen, C. Y.; Du, X.; Li, Y. M.; Lu, Z.-T.; O'Connor, T. P.; Young, L.



Effect of the ion distribution over longitudinal velocities on the efficiency and separating parameters of the ICR method of isotope separation  

Microsoft Academic Search

In the context of the ICR method of isotope separation, resonance RF heating of the ions in an electric field propagating\\u000a along a constant magnetic field while simultaneously rotating in the direction perpendicular to it is calculated in a linear\\u000a approximation. The analysis is carried out for two types of the initial ion distribution function over longitudinal velocities:\\u000a a function

E. P. Potanin



Isotope spectroscopy  

NASA Astrophysics Data System (ADS)

The measurement of isotopic ratios provides a privileged insight both into nucleosynthesis and into the mechanisms operating in stellar envelopes, such as gravitational settling. In this article, we give a few examples of how isotopic ratios can be determined from high-resolution, high-quality stellar spectra. We consider examples of the lightest elements, H and He, for which the isotopic shifts are very large and easily measurable, and examples of heavier elements for which the determination of isotopic ratios is more difficult. The presence of 6Li in the stellar atmospheres causes a subtle extra depression in the red wing of the 7Li 670.7 nm doublet which can only be detected in spectra of the highest quality. But even with the best spectra, the derived 6Li abundance can only be as good as the synthetic spectra used for their interpretation. It is now known that 3D non-LTE modelling of the lithium spectral line profiles is necessary to account properly for the intrinsic line asymmetry, which is produced by convective flows in the atmospheres of cool stars, and can mimic the presence of 6Li. We also discuss briefly the case of the carbon isotopic ratio in metal-poor stars, and provide a new determination of the nickel isotopic ratios in the solar atmosphere.

Caffau, E.; Steffen, M.; Bonifacio, P.; Ludwig, H.-G.; Monaco, L.; Lo Curto, G.; Kamp, I.



Semisupervised learning using negative labels.  


The problem of semisupervised learning has aroused considerable research interests in the past few years. Most of these methods aim to learn from a partially labeled dataset, i.e., they assume that the exact labels of some data are already known. In this paper, we propose to use a novel type of supervision information to guide the process of semisupervised learning, which indicates whether a point does not belong to a specific category. We call this kind of information negative label (NL) and propose a novel approach called NL propagation (NLP) to efficiently make use of this type of information to assist the process of semisupervised learning. Specifically, NLP assumes that nearby points should have similar class indicators. The data labels are propagated under the guidance of NL information and the geometric structure revealed by both labeled and unlabeled points, by employing some specified initialization and parameter matrices. The convergence analysis, out-of-sample extension, parameter determination, computational complexity, and relations to other approaches are presented. We also interpret the proposed approach within the framework of regularization. Promising experimental results on image, digit, spoken letter, and text classification tasks are provided to show the effectiveness of our method. PMID:21233049

Hou, Chenping; Nie, Feiping; Wang, Fei; Zhang, Changshui; Wu, Yi



Stable isotope analysis of breath using the optogalvanic effect  

Microsoft Academic Search

A new technique based on the optogalvanic effect has been developed for the measurement of stable isotope ratios in the carbon dioxide of exhaled breath. Data obtained before and after ingestion of harmless stable isotope labeled compounds, metabolized to carbon dioxide, can be used for sensitive noninvasive diagnostics of various disease conditions. The technique uses the specificity of laser resonance

Daniel E. Murnick; M. J. Colgan; H. P. Lie; D. Stoneback



Probing label-free intracellular quantification of free peptide by MALDI-ToF mass spectrometry.  


Cell-penetrating peptides are promising reagents for gene and drug delivery. They can efficiently traverse the plasma membrane and deliver various cargo materials ranging from genes to nanoparticles. The functional efficiency of cargo often depends on the completeness of intracellular peptide uptake, which can be measured, but its quantification remains largely inconclusive. Existing approaches rely on the use of radioactive and fluorescent labels or tags which allow colorimetric, fluorescent or spectrometric detection, but lack the ability to detect free peptide. Herein we describe a generic label- and tag-free method to measure the concentration of internalised peptide by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Quantification is preceded by two-dimensional chromatography and is performed at benign temperatures for the lysates of human dermal fibroblasts transfected with cell penetrating peptides in free form. Isotopically labelled peptides of the same structure are used as internal standards to enable accurate determination of concentration of the recovered free peptide. The method offers a minimalistic approach for intracellular quantification, which can be used as a correlative measure for fluorescence-based imaging methods. PMID:24657280

Rakowska, Paulina D; Lamarre, Baptiste; Ryadnov, Maxim G



Model-shared subspace boosting for multi-label classification  

Microsoft Academic Search

Typical approaches to the multi-label classification problem require learning an independent classifier for every label from all the examples and features. This can become a computational bottleneck for sizeable datasets with a large label space. In this paper, we propose an efficient and effective multi-label learning algorithm called model-shared subspace boosting (MSSBoost) as an attempt to reduce the information redundancy

Rong Yan; Jelena Tesic; John R. Smith



Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry  

PubMed Central

Multi-isotope imaging mass spectrometry (MIMS) is the quantitative imaging of stable isotope labels in cells with a new type of secondary ion mass spectrometer (NanoSIMS). The power of the methodology is attributable to (i) the immense advantage of using non-toxic stable isotope labels, (ii) high resolution imaging that approaches the resolution of usual transmission electron microscopy and (iii) the precise quantification of label down to 1 part-per-million and spanning several orders of magnitude. Here we review the basic elements of MIMS and describe new applications of MIMS to the quantitative study of metabolic processes including protein and nucleic acid synthesis in model organisms ranging from microbes to humans. PMID:23660233

Steinhauser, Matthew L.; Lechene, Claude P.



Assessment of vitamin A status in rats by isotope dilution: A simplified model  

SciTech Connect

Isotope-dilution analysis of vitamin A status requires giving a known quantity of labeled vitamin A to the subject and measuring the ratio of labeled to unlabeled retinol in the blood after a period for equilibration. To calculate total body stores from the isotopic ratio of plasma retinol, several assumptions must be made. In considering new ways of better calculating liver vitamin A stores from isotope-dilution data, the authors used the data of Green et al. to estimate loss of vitamin A tracer as a function of time and of vitamin A status. This correction markedly improves the correlation between calculated and analyzed liver vitamin A stores and also quantitively explains the hyperbolic relationship between fraction of tracer dose recovered in liver and mass of liver vitamin A stores. Agreement of this model with experimental data suggests that efficiency of absorption and storage of vitamin A is not affected by vitamin A status. This model can be used to estimate both the amount of tracer needed for a given lower limit of detection and an optimum sampling time.

Furr, H.C.; Cooper, D.A.; Olson, J.A. (Iowa State Univ., Ames (United States))



Isolation and optimisation of the oleaginous yeast Sporobolomyces roseus for biosynthesis of 13C isotopically labelled 18-carbon unsaturated fatty acids and trans 18:1 and 18:2 derivatives through synthesis.  


An oleaginous and psychrotrophic strain (F38-3) of Sporobolomyces roseus Kluyver & van Niel was isolated from a salt marsh environment in Nova Scotia, Canada following a screening program to select for high producers of 18-carbon unsaturated fatty acids. Fatty acid production was characterised as a function of temperature at 20 g glucose L(-1), and optimal yields were obtained at 14°C, achieving 5.7 g dw biomass and 39.2% total fatty acids by dry weight, with 18:1, 18:2 and 18:3 all-cis fatty acids accounting for 49.4%, 14.3% and 6.7% of total fatty acids (TFA), respectively--the highest reported for this species. Production of 18:3 was inversely correlated to growth temperature, rising from 2% of TFA at 30°C to 8.9% at 6°C. Cultivation of isolate F38-3 on universally (13)C (U-(13)C) labelled glucose and subsequent transesterification and isolation of the fatty acid methyl esters (FAMEs) by preparative chromatography yielded pure, highly (13)C-enriched (>90%) 18:1, 18:2 and 18:3 all-cis FAMEs. The U-(13)C 18:1 FAME was catalytically converted to U-(13)C 18:1 trans-9 and purified to >99.5% purity. The U-(13)C 18:2 was converted by alkaline isomerisation into a 50/50 mixture of 18:2 cis-9, trans-11 and 18:2 trans-10, cis-12 isomers and purified to >95.0% purity. Overall, 10%, by weight, of labelled glucose fed to isolate F38-3 was recovered as fatty acid methyl esters and 7.5% as 18-carbon unsaturated fats, and the final isomerisation reactions resulted in yields of 80% or greater. The ultimate goal of the work is to develop methodologies to produce (13)C-labelled metabolic tracers as tools to study the metabolism of trans fats. PMID:21809096

Cui, Yi; Fraser, Catharine; Gardner, Graeme; Huang, Ching-Jang; Reith, Michael; Windust, Anthony J



Mental Labels and Tattoos  

ERIC Educational Resources Information Center

Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

Hyatt, I. Ralph



Native SILAC: metabolic labeling of proteins in prototroph microorganisms based on lysine synthesis regulation.  


Mass spectrometry (MS)-based quantitative proteomics has matured into a methodology able to detect and quantitate essentially all proteins of model microorganisms, allowing for unprecedented depth in systematic protein analyses. The most accurate quantitation approaches currently require lysine auxotrophic strains, which precludes analysis of most existing mutants, strain collections, or commercially important strains (e.g. those used for brewing or for the biotechnological production of metabolites). Here, we used MS-based proteomics to determine the global response of prototrophic yeast and bacteria to exogenous lysine. Unexpectedly, down-regulation of lysine synthesis in the presence of exogenous lysine is achieved via different mechanisms in different yeast strains. In each case, however, lysine in the medium down-regulates its biosynthesis, allowing for metabolic proteome labeling with heavy-isotope-containing lysine. This strategy of native stable isotope labeling by amino acids in cell culture (nSILAC) overcomes the limitations of previous approaches and can be used for the efficient production of protein standards for absolute SILAC quantitation in model microorganisms. As proof of principle, we have used nSILAC to globally analyze yeast proteome changes during salt stress. PMID:23592334

Fröhlich, Florian; Christiano, Romain; Walther, Tobias C



Lead isotopes in environmental sciences: A review  

Microsoft Academic Search

Lead (Pb) isotopic analyses proved to be a very efficient tool for tracing the sources of local and global Pb pollution. This review presents an overview of literature published on the use of Pb isotopic analyses of different environmental matrices (atmospheric aerosols, lichens, tree rings, peat deposits, lake, stream, marine sediments, soils, etc.). In order to gain more insight, the

Michael Komárek; Vojt?ch Ettler; Vladislav Chrastný; Martin Mihaljevi?



Isotopically nonstationary MFA (INST-MFA) of autotrophic metabolism.  


Metabolic flux analysis (MFA) is a powerful approach for quantifying plant central carbon metabolism based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstationary (13)C MFA (INST-MFA), which is applicable to autotrophic systems that are at metabolic steady state but are sampled during the transient period prior to achieving isotopic steady state following the introduction of (13)CO2. We describe protocols for performing the necessary isotope labeling experiments, sample collection and quenching, nonaqueous fractionation and extraction of intracellular metabolites, and mass spectrometry (MS) analysis of metabolite labeling. We also outline the steps required to perform computational flux estimation using INST-MFA. By combining several recently developed experimental and computational techniques, INST-MFA provides an important new platform for mapping carbon fluxes that is especially applicable to autotrophic organisms, which are not amenable to steady-state (13)C MFA experiments. PMID:24222417

Jazmin, Lara J; O'Grady, John P; Ma, Fangfang; Allen, Doug K; Morgan, John A; Young, Jamey D



26Mg labeling of the sea urchin regenerating spine: Insights into echinoderm biomineralization process  

Microsoft Academic Search

This paper reports the results of the first dynamic labeling experiment with regenerating spines of sea urchins Paracentrotus lividus using the stable isotope 26Mg and NanoSIMS high-resolution isotopic imaging, which provide a direct information about the growth process. Growing spines were labeled twice (for 72 and 24h, respectively) by increasing the abundance of 26Mg in seawater. The incorporation of 26Mg

Przemys?aw Gorzelak; Jaros?aw Stolarski; Philippe Dubois; Christophe Kopp; Anders Meibom



Towards a "perfect" Penning trap mass spectrometer for unstable isotopes  

NASA Astrophysics Data System (ADS)

A Penning trap mass spectrometer has been set up at the on-line isotope separator ISOLDE/CERN for the mass determination of unstable heavy isotopes. The spectrometer should fulfil the following requirements: capture of external ions in high efficiency, high resolving power and accuracy, general applicability to all elements and isotopes available at the on-line facility.

Bollen, G.; Hartmann, H.; Kluge, H.-J.; König, M.; Otto, T.; Savard, G.; Stolzenberg, H.; ISOLDE Collaboration



Oxygen isotope evolution in the protosolar disk induced by isotopic exchange reactions  

NASA Astrophysics Data System (ADS)

Oxygen isotopic heterogeneity is one of the most extensively studied and debated topics in planetary science, though no consensus has been achieved yet. We quantitatively examine the role of exchange reactions in mass-dependent oxygen isotope fractionation during evaporation and recondensation of silicate melts in gas with isotopically identical and distinct gases. The results are applied to the CCAM line, mass-dependent oxygen isotopic fractionation of FUN inclusions, and oxygen isotopic variations of Allende chondrules. We examine the role of oxygen isotopic exchange for silicate melt and ambient gas during evaporation and recondensation in a closed system that monotonically cools from high temperature. Silicate dust aggregates are assumed to be instantaneously heated to an above liquidus temperature. There are three free parameters; one is the efficiency of the isotope ex-change, the second is the cooling rate of the system, and the third is the amount of initial oxygen in the ambient gas. We found following results : (1) Evolution of oxygen isotopic fractionation is decoupled with chemical fractionation of silicate melt; oxygen isotopic fractionation goes more rapidly than chemical fractionation, of which timing depends on the isotopic exchange efficiency. (2) Mass-dependent oxygen isotopic fractionation of silicate melt is suppressed by isotopic exchange during evaporation/recondensation. If isotopic exchange does not work, oxygen isotopic composition of silicate melt spheres after evaporation / recondensation becomes lighter than the initial one if the silicate and gas have identical oxygen isotopic composition due to more abundance of 16O in the gas. Isotopic exchange homogenizes the compositions effectively to result in the same composition between silicate and gas. (3) Cooling rate of the system affects the evolution time of oxygen isotopic mass fractionation, but the final composition is not affected. In a system with initially different oxygen isotopic compositions between silicate melt and gas, deviation from a straight mixing line to the ?18O-rich side on a three-oxygen isotope plot is inevitable through evaporation/recondensation and isotope exchange. (4) Abundance of oxygen in the initial gas largely affects the degree of deviation from a simple mixing line during the cooling process. The average oxygen abundance in gas of protosolar disk relative to that in silicate (~10), and silicate melt gets oxygen isotopic composition very close to the gas. (5) Isotopic exchange efficiency plays an important role on evolution and final oxygen isotopic composition of silicate melt heated in gas. Effective isotopic exchange largely suppresses mass dependent isotopoic fractionation, which tends to result in forming a straight "mixing line", whereas, smaller efficiency causes larger degree of mass-dependent isotopic fractionation, and deviation from a straight "mixing line" becomes larger. (6) A straight mixing line such as CCAM line requires the presence of oxygen in the ambient gas more than the average protosolar disk. (7) The mass-dependent oxygen isotope fractionation of FUN inclusions by ~40‰ or more is not achieved by evaporation in a closed system, and almost vacuum condition is required, which was possible by almost complete continuous separation of gas and silicate melt during evaporation. If FUN inclusions were formed from oxygen isotopically light solid, they have suffered a reaction between isotopically heavy gas after vacuum evaporation, where the gas abundance must have been more than 10 times of the average protosolar gas. In summary, a series of oxygen isotopic composition of CAIs and chondrules reflects the ambient physical conditions such as dust/gas ratio and dust/gas separation of a protosolar disk.

Nagahara, H.



Isotope-coded carbamidomethylation for quantification of N-glycoproteins with online microbore hollow fiber enzyme reactor-nanoflow liquid chromatography-tandem mass spectrometry.  


This paper introduces a simple, inexpensive, and robust quantitative proteomic method for quantifying N-linked glycoproteins based on isotope-coded carbamidomethylation (iCCM) incorporated into an online microbore hollow fiber enzyme reactor and nanoflow liquid chromatography-tandem mass spectrometry (mHFER-nLC-MS/MS). The iCCM quantitation uses carbamidomethylation (CM; a routine protection of thiol groups before proteolysis) of the Cys residue of proteins with iodoacetamide (IAA) or its isotope (IAA-(13)C2,D2: 4 Da difference). CM-/iCCM-labeled proteome samples are mixed for proteolysis; then, online enrichment of N-glycopeptides using lectin affinity is carried out in an mHFER before nLC-MS/MS for quantification using multiple reaction monitoring (MRM). Initial evaluation of the iCCM method varying the mixing ratio of CM-/iCCM-labeled bovine serum albumin (BSA) standards yielded successful quantification of 18 peptides with less than 2% variation in the calculated ratio of light/heavy-labeled peptides. The iCCM quantitation with mHFER-nLC-MS/MS was evaluated with three standard glycoproteins (?-1-acid glycoproteins, fetuin and transferrin) and then applied to serum glycoproteins from liver cancer patients and controls, resulting in successful quantification of 73 N-glycopeptides (from 49 N-glycoproteins), among which 19 N-glycopeptides from 14 N-glycoproteins showed more than a 2.5-fold aberrant change in liver cancer patients' sera compared with the pooled control. Although iCCM quantitation with mHFER-nLC-MS/MS applies only to glycopeptides with Cys residue, the method can offer several advantages over other labeling methods when applied to targeted glycoproteins: The iCCM method does not require an additional labeling reaction under special conditions nor complicated procedures to purify labeled products using additional columns. Isotope labeling at the protein level can minimize potential uncertainty originating from unequal efficiencies in protein digestion in separate vials and retrieval of each labeled peptide when labeling takes place at the peptide level. In addition, the labeling reagents for the iCCM method are readily obtained at a reasonable cost, which can make protein quantification easily accessible. PMID:24960276

Kim, Jin Yong; Oh, Donggeun; Kim, Sook-Kyung; Kang, Dukjin; Moon, Myeong Hee



Labeling earthworms uniformly with 13C and 15N: implications for monitoring nutrient fluxes  

Microsoft Academic Search

Stable isotopes hold promise for improving our ability to quantify energy and N released from earthworm populations through metabolic processes and mortality. However, the isotopic labels 13C and 15N must be incorporated uniformly into the structural and labile tissues of earthworms to trace C and N fluxes accurately. We examined the distribution of 13C and 15N in the tissue and

Joann K Whalen; H. Henry Janzen



DNA Hybridization: Nonradioactive Labeling Now Available for the Laboratory.  

ERIC Educational Resources Information Center

The advantages of DNA hybridization procedures for classroom and clinical use can now be realized by the recent development of nonradioactive DNA labeling/detection procedures. These methods (which are described) can replace the use of isotopes in standard DNA hybridization procedures. (JN)

Freeman, Lenore Gardner



Mass Spectrometry-Based Label-Free Quantitative Proteomics  

PubMed Central

In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies. PMID:19911078

Zhu, Wenhong; Smith, Jeffrey W.; Huang, Chun-Ming



A new [(2) H]-labelled ?-trichloroimidate glucuronic ester for the synthesis of deuterated drug conjugates.  


A new reaction pathway for the synthesis of a [(2) H]-labelled trichloroacetimidate precursor for the preparation of glucuronides is described. Therewith, stable isotope-labelled drug glucuronides become accessible on a preparative scale, which can further be used as internal standards for quantitative analysis. PMID:25339577

Heinkele, Georg; Geditz, Mirjam C K; Ganchev, Boian; Kerb, Reinhold; Hofmann, Ute; Mürdter, Thomas E



Isotope separation apparatus and method  


The invention relates to an improved method and apparatus for laser isotope separation by photodeflection. A molecular beam comprising at least two isotopes to be separated intersects, preferably substantially perpendicular to one broad side of the molecular beam, with a laser beam traveling in a first direction. The laser beam is reflected back through the molecular beam, preferably in a second direction essentially opposite to the first direction. Because the molecules in the beam occupy various degenerate energy levels, if the laser beam comprises chirped pulses comprising selected wavelengths, the laser beam will very efficiently excite substantially all unexcited molecules and will cause stimulated emission of substantially all excited molecules of a selected one of the isotopes in the beam which such pulses encounter. Excitation caused by first direction chirped pulses moves molecules of the isotope excited thereby in the first direction. Stimulated emission of excited molecules of the isotope is brought about by returning chirped pulses traveling in the second direction. Stimulated emission moves emitting molecules in a direction opposite to the photon emitted. Because emitted photons travel in the second direction, emitting molecules move in the first direction. Substantial molecular movement of essentially all the molecules containing the one isotope is accomplished by a large number of chirped pulse-molecule interactions. A beam corer collects the molecules in the resulting enriched divergent portions of the beam.

Feldman, Barry J. (Los Alamos, NM)



International cigarette labelling practices  

Microsoft Academic Search

DESIGNCross-sectional study.PARTICIPANTSMembers of GLOBALink (, an internet listserve for tobacco activists with members in 56 countries, who were asked to provide specific information on cigarette warning requirements in their countries.MAIN OUTCOME MEASURESPresence of specific warning labels, overall content score (based on a 0–10 scale with a point for each specific warning mentioned), size of warning label, location of warning label.RESULTSForty-five

Macksood Aftab; Deborah Kolben; Peter Lurie



Isotopes in groundwater hydrology  

Microsoft Academic Search

Isotopes in groundwater hydrology give a direct insight into the movement and distribution processes within the aquifer. Groundwater in its natural state contains environmental isotopes and conclusions may be drawn from their abundance variations. The isotopes commonly employed in groundwater investigations are the heavy stable isotopes of the water molecule, deuterium and oxygen-18 and the radioactive isotopes, tritium and carbon-14.



Bar Code Labels  

NASA Technical Reports Server (NTRS)

American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.



Measurement of protein turnover rates by heavy water labeling of nonessential amino acids  

Microsoft Academic Search

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C–H bonds of nonessential AAs (NEAAs)

Robert Busch; Yoo-Kyeong Kim; Richard A. Neese; Valerie Schade-Serin; Michelle Collins; Mohamad Awada; James L. Gardner; Carine Beysen; Michael E. Marino; Lisa M. Misell; Marc K. Hellerstein



Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short-strong hydrogen bonded cluster  

E-print Network

Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short/deuterium isotope effects, in a fundamental organic hydrogen bonded system using multiple experimental infrared the isotopically labeled systems arises from an analysis of the simulated cluster spectroscopy and leads

Iyengar, Srinivasan S.


Isotope effect of the phonons mean free path in graphene by micro-Raman measurement  

NASA Astrophysics Data System (ADS)

The isotope labeled graphene was synthesized in the concentration of 13C carbon atom in 1%, 25%, 50%, 75% and 99%. The isotope effect on the phonon behavior in graphene was investigated based on the micro-Raman analysis of 13C isotope labeled graphene samples. We found that the phonon scattering is affected by the isotopic carbon atom as a point defect. Based on the experiment results, the Klemens-Callaway model and uncertainty principle were used to obtain the mean free path of the G and D phonons. The results agree with the thermal conductivity measurement by non-contact optical method and with other theoretical calculations.

Zhang, CanKun; Li, QiongYu; Tian, Bo; Huang, ZhiYi; Lin, WeiYi; Li, HongYang; He, DaHai; Zhou, YingHui; Cai, WeiWei



Tritium labeling of detonation nanodiamonds.  


For the first time, the radioactive labeling of detonation nanodiamonds was efficiently achieved using a tritium microwave plasma. According to our measurements, the total radioactivity reaches 9120 ± 120 ?Ci mg(-1), with 93% of (3)H atoms tightly bonded to the surface and up to 7% embedded into the diamond core. Such (3)H doping will ensure highly stable radiolabeled nanodiamonds, on which surface functionalization is still allowed. This breakthrough opens the way to biodistribution and pharmacokinetics studies of nanodiamonds, while this approach can be scalable to easily treat bulk quantities of nanodiamonds at low cost. PMID:24492594

Girard, Hugues A; El-Kharbachi, Abdelouahab; Garcia-Argote, Sébastien; Petit, Tristan; Bergonzo, Philippe; Rousseau, Bernard; Arnault, Jean-Charles



Label Review Manual.  

National Technical Information Service (NTIS)

The Labeling Center for Excellence (LCE) within the Office of Pesticide Programs (OPP) developed this manual to serve as a training tool for new employees and as a source of direction for product team members who are responsible for performing label revie...

M. Waller, M. Johnson, T. Adamczyk, J. Downing



Using Food Labels  

NSDL National Science Digital Library

In this nutrition activity, learners explore food labels and consider the nutritional value of foods. Learners also explore units of measurement commonly used on food labels. Learners will be surprised to find out how much sugar soft drinks contain. This lesson guide includes background information and bilingual (English/Spanish) handouts.

Moreno, Nancy P.; Tharp, Barbara Z.



Assessment of the production of 13C labeled compounds from phototrophic microalgae at laboratory scale.  


An integrated process for the indoor production of 13C labeled polyunsaturated fatty acids (PUFAs) from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor operating with recirculation of the exhaust gas using a low-pressure compressor. Oxygen accumulation in the system is avoided by bubbling the exhaust gas from the reactor in a sodium sulfite solution before returning to it. To achieve a high 13C enrichment in the biomass obtained, the culture medium is initially stripped of carbon, and labeled 13CO(2) is automatically injected on-demand during operation for pH control and carbon supply. The reactor was operated in both batch and semicontinuous modes. In semicontinuous mode, the reactor was operated at a dilution rate of 0.01 h(-1), resulting in a biomass productivity of 0.1 g l(-1) per day. The elemental analysis of the inlet and outlet flows of the reactor showed that 64.9% of carbon was turned into microalgal biomass, 34.9% remained in the supernatant mainly as inorganic compounds. Only 3.8% of injected carbon was effectively fixed as the target labeled product (EPA). Regarding the isotopic composition of fatty acids, results showed that fatty acids were not labeled in the same proportion, the higher the number of carbons the lower the percentage of 13C. Isotopic composition of EPA ranged from 36.5 to 53.5%, as a function of the methodology used (GC-MS, EA-IRMS or gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS)). The low carbon uptake efficiency combined with the high cost of 13CO(2) make necessary to redefine the designed culture system to increase the efficiency of the conversion of 13CO(2) into the target product. Therefore, the possibility of removing 12C from the fresh medium, and recovering and recirculating the inorganic carbon in the supernatant and the organic carbon from the EPA depleted biomass was studied. The inorganic carbon of the fresh medium was removed by acidification and stripping with N(2). The inorganic carbon of the supernatant was recovered also by acidification and subsequent stripping with N(2). The operating conditions of this step were optimized for gas flow rate and type of contactor. A carbon recovery step for the depleted biomass was designed based on the catalytic oxidation to CO(2) using CuO (10 wt.%) as catalyst with an oxygen enriched atmosphere (80% O(2) partial pressure). In this way, the carbon losses reduced an 80.2% and the efficiency of the conversion of carbon in EPA was increased to 19.5%, which is close to the theoretical maximum. Further increase in 13CO(2) use efficiency is only possible by additionally recovering other labeled by-products present in the biomass: proteins, carbohydrates, lipids, and pigments. PMID:12919792

Acién Fernández, Francisco G; Alías, Celeste Brindley; García-Malea López, María C; Fernández Sevilla, José M; Ibáñez González, María J; Gómez, Rafael Núñez; Molina Grima, Emilio



Investigation of the reaction pathways in selective catalytic reduction of NO with NH{sub 3} over V{sub 2}O{sub 5} catalysts: Isotopic labeling studies using {sup 18}O{sub 2}, {sup 15}NH{sub 3}, {sup 15}NO, and {sup 15}N{sup 18}O{sup 1}  

SciTech Connect

Isotopic tracer studies were performed to investigate the reaction network of selective catalytic reduction of nitric oxide over vanadia catalysts having preferential exposure of different crystal planes. The catalysts were characterized using BET surface area analysis, X-ray diffraction, laser Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, 3-D imaging, and thermal analysis techniques. The product analysis was carried out by a combination of chemiluminescence NO{sub x} analysis, gas chromatography-mass spectrometry, and chemical titration methods. The isotropic labeling experiments were performed under steady state reaction conditions by using NH{sub 3} + NO + {sup 16}O{sub 2} {r_arrow} NH{sub 3} + NO + {sup 18}O{sub 2}, {sup 14}NH{sub 3} + NO + O{sub 2} {r_arrow} {sup 15}NH{sub 3} + NO + O{sub 2}, NH{sub 3} + {sup 14}NO + O{sub 2} {r_arrow} NH{sub 3} + {sup 15}NO + O{sub 2}, and NH{sub 3} + {sup 14}N{sup 16}O + {sup 16}O{sub 2} {r_arrow} NH{sub 3} + {sup 15}N{sub 18}O + {sup 18}O{sub 2} switches. Interaction of NO with the vanadia surface was also studied using a {sup 14}N{sup 16}O {r_arrow} {sup 15}N{sup 18}O switch. The results obtained in these experiments were combined with the tracer studies performed in ammonia oxidation reactions over the same catalysts to elucidate the reaction pathways involved in SCR reactions, to compare the surface residence times of nitrogen containing species, as well as to quantify the role of ammonia oxidation in SCR reactions. 35 refs., 14 figs., 3 tabs.

Ozkan, U.S.; Cai, Y.; Kumthekar, M.W. [Ohio State Univ., Columbus, OH (United States)] [Ohio State Univ., Columbus, OH (United States)



Noise labeling in Brazil  

NASA Astrophysics Data System (ADS)

The Brazilian Silence Program, created in 1990 by the Brazilian Ministry of Environment, advocates the production and use of equipment with lower noise level. The subcommittee of Noise Labeling of the Brazilian Committee of Certification is composed of INMETRO acoustic specialists to organize and implement the Brazilian Labeling Program. This subcommittee elaborated the label form and test procedure. The noise-labeling program will first concentrate on the following household devices, both manufactured in Brazil or imported from abroad; mixers, blenders, hairdryers, refrigerators, and vacuum cleaners. The label should contain the sound-power level in dBA. INMETRO or other credited laboratories are responsible for the measurements. The ISO 4871, 3740 (1 to 5), ISO 8960, and IEC 704 (1 to 4) and also the equivalent Brazilian standards are used for the measurements, such as ABNT NBR 13910-1. The main objective of the label is to inform the consumer about the emitted noise level. The label offers the noise parameter to be used by the consumer when comparing devices, considering price, performance, and now also noise. No restriction for noise level was established.

de Araujo, Marco A. N.; Massarani, Paulo M.; de Azevedo, Jose A. J.; Gerges, Samir N. Y.



Conifers, Angiosperm Trees, and Lianas: Growth, Whole-Plant Water and Nitrogen Use Efficiency, and Stable Isotope Composition (?13C and ?18O) of Seedlings Grown in a Tropical Environment1[W][OA  

PubMed Central

Seedlings of several species of gymnosperm trees, angiosperm trees, and angiosperm lianas were grown under tropical field conditions in the Republic of Panama; physiological processes controlling plant C and water fluxes were assessed across this functionally diverse range of species. Relative growth rate, r, was primarily controlled by the ratio of leaf area to plant mass, of which specific leaf area was a key component. Instantaneous photosynthesis, when expressed on a leaf-mass basis, explained 69% of variation in r (P < 0.0001, n = 94). Mean r of angiosperms was significantly higher than that of the gymnosperms; within angiosperms, mean r of lianas was higher than that of trees. Whole-plant nitrogen use efficiency was also significantly higher in angiosperm than in gymnosperm species, and was primarily controlled by the rate of photosynthesis for a given amount of leaf nitrogen. Whole-plant water use efficiency, TEc, varied significantly among species, and was primarily controlled by ci/ca, the ratio of intercellular to ambient CO2 partial pressures during photosynthesis. Instantaneous measurements of ci/ca explained 51% of variation in TEc (P < 0.0001, n = 94). Whole-plant 13C discrimination also varied significantly as a function of ci/ca (R2 = 0.57, P < 0.0001, n = 94), and was, accordingly, a good predictor of TEc. The 18O enrichment of stem dry matter was primarily controlled by the predicted 18O enrichment of evaporative sites within leaves (R2 = 0.61, P < 0.0001, n = 94), with some residual variation explained by mean transpiration rate. Measurements of carbon and oxygen stable isotope ratios could provide a useful means of parameterizing physiological models of tropical forest trees. PMID:18599645

Cernusak, Lucas A.; Winter, Klaus; Aranda, Jorge; Turner, Benjamin L.



How to Read Drug Labels  


... and alternative medicine Healthy Aging How to read drug labels Printer-friendly version How to Read Drug ... read drug labels How to read a prescription drug label View a text version of this picture. ...


Comprehensive analysis of metabolic pathways through the combined use of multiple isotopic tracers  

E-print Network

Metabolic Flux Analysis (MFA) has emerged as a tool of great significance for metabolic engineering and the analysis of human metabolic diseases. An important limitation of MFA, as carried out via stable isotope labeling ...

Antoniewicz, Maciek Robert



Informational Aspects of Isotopic Diversity in Biology and Medicine  

NASA Astrophysics Data System (ADS)

Use of stable and radioactive isotopes in biology and medicine is intensive, yet informational aspects of isotopes as such are largely neglected (A.A.Berezin, J.Theor.Biol.,1992). Classical distinguishability (``labelability'') of isotopes allows for pattern generation dynamics. Quantum mechanically advantages of isotopicity (diversity of stable isotopes) arise from (almost perfect) degeneracy of various isotopic configurations; this in turn allows for isotopic sweeps (hoppings) by resonance neutron tunneling (Eccles mechanism). Isotopic variations of de Broglie wavelength affect quantum tunneling, diffusivity, magnetic interactions (e.g. by Lorentz force), etc. Ergodicity principle (all isoenergetic states are eventually accessed) implies possibility of fast scanning of library of morphogenetic patterns (cf metaphors of universal ``Platonic'' Library of Patterns: e.g. J.L.Borges, R.Sheldrake) with subsequent Darwinian reinforcement (e.g. by targeted mutations) of evolutionary advantageous patterns and structures. Isotopic shifts in organisms, from viruses and protozoa to mammalians, (e.g. DNA with enriched or depleted C-13) are tools to elucidate possible informational (e.g. Shannon entropy) role of isotopicity in genetic (e.g. evolutionary and morphological), dynamical (e.g. physiological and neurological) as well as medical (e.g. carcinogenesis, aging) aspects of biology and medicine.

Berezin, Alexander A.



Stable isotope studies  

SciTech Connect

The research has been in four general areas: (1) correlation of isotope effects with molecular forces and molecular structures, (2) correlation of zero-point energy and its isotope effects with molecular structure and molecular forces, (3) vapor pressure isotope effects, and (4) fractionation of stable isotopes. 73 refs, 38 figs, 29 tabs.

Ishida, T.



Like your labels?  


The descriptive “conventions” used on food labels are always evolving. Today, however, the changes are so complicated (partly driven by legislation requiring disclosures about environmental impacts, health issues, and geographical provenance) that these labels more often baffle buyers than enlighten them. In a light-handed manner, the article points to how sometimes reading label language can be like deciphering runes—and how if we are familiar with the technical terms, we can find a literal meaning, but still not see the implications. The article could be ten times longer because food labels vary according to cultures—but all food-exporting cultures now take advantage of our short attention-span when faced with these texts. The question is whether less is more—and if so, in this contest for our attention, what “contestant” is voted off. PMID:21539053

Field, Michele



Spectral Label Fusion  

E-print Network

We present a new segmentation approach that combines the strengths of label fusion and spectral clustering. The result is an atlas-based segmentation method guided by contour and texture cues in the test image. This offers ...

Wachinger, Christian


Food Label and You  

MedlinePLUS Videos and Cool Tools

... Restaurants & Retail Establishments The Food Label and You — Video FDA presents an entertaining and educational tool to ... informed food choices. You can view the new video in its entirety or select on any of ...



EPA Science Inventory

Carbon isotope ratios ( 13C) of tree rings are commonly used for paleoclimatic reconstruction and for inferring canopy water-use efficiency (WUE). However, the responsiveness of carbon isotope discrimination ( ) to site disturbance and resource availability has only rarely been ...


An inexact labeling problem  

E-print Network

AH IHEXACT LABELING PROBLEM A Thesis Margaret Shuey Murray Submitted to the Graduate College of Texas ASM University in partial fulfillment of the requirements for tbs degree of MASTER OF SCIEHCE May 1984 Major Subject: Electrical... Engineering AN INEXACT LABELING PROBLEM A Thesis Margaret Shuey Murray Approved as to style and content by: Norman Griswol (Chairman of Cosssi tee) Jo Howze (Member) Graham Allen (Member) for William B. Jones Jr. (Head of Department) May 1984...

Murray, Margaret Shuey



Photosynthetic carbon isotope discrimination and its relationship to the carbon isotope signals of stem, soil and ecosystem respiration (Invited)  

NASA Astrophysics Data System (ADS)

Photosynthetic carbon (C) isotope discrimination labels photosynthates (?A) and atmospheric CO2 (?a) with variable C isotope compositions during fluctuating environmental conditions. In this context, the C isotope composition of respired CO2 within ecosystems is often hypothesized to vary temporally with photosynthetic discrimination. We investigated the relationship between photosynthetic discrimination and the C isotope signals from stem (?W), soil (?S) and ecosystem (?E) respired CO2 to environmental fluctuations, using novel tuneable diode laser absorption spectrometer instrumentation in a mature maritime pine forest. Broad seasonal changes in photosynthetic discrimination were reflected in ?W, ?S and ?E. However, respired CO2 signals had smaller short-term variations than photosynthetic discrimination and were offset and delayed by 2-10 d, indicating fractionation and isotopic mixing in a large C pool. Variations in ?S did not follow photosynthetic discrimination at all times, especially during rainy periods and when there is a strong demand for C allocation above ground. It is likely that future isotope-enabled vegetation models will need to develop transfer functions that can account for these phenomena in order to interpret and predict the isotopic impact of biosphere gas exchange on the C isotope composition of atmospheric CO2. L. Wingate, J. Ogée, R. Burlett, A. Bosc, M. Devaux, J. Grace, D. Loustau and A. Gessler. Photosynthetic carbon isotope discrimination and its relationship to the carbon isotope signals of stem, soil and ecosystem respiration. New Phytologist, doi: 10.1111/j.1469-8137.2010.03384.x

Wingate, L.; Ogée, J.; Burlett, R.; Bosc, A.; Devaux, M.; Grace, J.; Loustau, D.; Gessler, A.



Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels.  


Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3' or 5' end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3' end and an electrochemical label at 5' end. PMID:23324407

Moreau, Julie; Dendane, Nabil; Schöllhorn, Bernd; Spinelli, Nicolas; Fave, Claire; Defrancq, Eric



Guide to good practices for equipment and piping labeling  

SciTech Connect

This Guide to Good Practices is written to enhance understanding of, and provide direction for, Equipment and Piping Labeling, Chapter XVIII of Department of Energy (DOE) Order 5480.19, Conduct of Operations Requirements for DOE Facilities. The practices in this guide should be considered when planning or reviewing labeling programs. Contractors are advised to adopt procedures that meet the intent of DOE Order 5480.19. Equipment and Piping Labeling is an element of an effective Conduct of Operations program. The complexity and array of activities performed in DOE facilities dictate the necessity for a coordinated labeling program to promote safe and efficient operations.




Fluorescent labels and their use in separations  


Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids.

Mathies, Richard A. (El Cerrito, CA); Glazer, Alexander (Orinda, CA); Ju, Jingyue (Berkeley, CA)



Probes labelled with energy transfer coupled dyes  


Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids.

Mathies, Richard A. (El Cerrito, CA); Glazer, Alexander (Orinda, CA); Ju, Jingyue (Berkeley, CA)



Hardware acceleration based connected component labeling algorithm in real-time ATR system  

NASA Astrophysics Data System (ADS)

Aims at the requirement of real-time processing in Real-Time Automatic Target Recognition(RTATR) system, this paper presents a hardware acceleration based two-scan connected-component labeling algorithm. Conventional pixel and run based algorithm's merits are combined, in the first scan, the pixel is processed scan unit while line as label unit, label equivalences are recorded while scanning the image by pixel. Lines with provisional label are outputted as the connected component labeling result. Then the union-find algorithm is used for resolving label equivalences and finds the representative label for each provisional label after the first scan. The labels are replaced in the second scan to complete the connected-component labeling. Experiments on RTATR platform demonstrate that the hardware acceleration implementation of algorithm reaches a higher performance and efficiency and consumes few resources. The implementation of proposed algorithm can meet the demand of real-time processing, and possesses a better practicability.

Zhao, Fei; Zhang, Zhi-yong



Embryotoxicity of stable isotopes and use of stable isotopes in studies of teratogenetic mechanisms  

SciTech Connect

Experiments on teratogenic effects of stable isotopes from our own and other laboratories are evaluated. In the first series of investigations, the enrichment of the stable isotope /sup 13/C derived from U-/sup 13/C-glucose was studied in mouse embryos at various stages of development, including limb buds in organ culture. Preimplantation mouse embryos incubated in vitro in /sup 13/C-enriched medium for 48 hours showed normal development during subsequent differentiation in vitro and also in vivo after embryo transfer to faster mothers. These embryos were 15% to 20% enriched in /sup 13/C. Administration of U-13-C-glucose to pregnant mice during organogenesis led to an increase of the absolute /sup 13/C content of the embryo for several days after the end of isotope administration, whereas the enrichment in maternal tissue decreased. No alterations of embryonic development were detected due to stable isotope enrichment. Development of cultured mouse limb buds was unaffected by incubation with 82 mol% U-/sup 13/C-glucose as judged from morphologic and biochemical criteria. The second part of the article describes the value of deuterium-labeled drugs as probes into the mechanism of activation of teratogenic metabolites. A comparison of the pharmacokinetics as well as the teratogenicity between cyclophosphamide and some specific deuterium-labeled analogues showed that the isotope effect observed can be related to a particular metabolic pathway crucial for teratogenic activation by this drug.

Spielmann, H.; Nau, H.



Transfer of Labelled Nitrogen from Glutamic Acid to Pine Seedlings through the Mycelium of Boletus variegatus (Sw.) Fr  

Microsoft Academic Search

IN a previous paper from this Institute1 it was shown, by using the isotope technique, that hyphæ of Boletus variegatus (Sw.) Fr. in mycorrhizal connexion with pine seedlings transfer nitrogen from a solution of an ammonium salt to the roots. The labelled nitrogen was added as ammonium nitrate containing the stable isotope nitrogen-15 in the ammonium group. By a special

Elias Melin; Harald Nilsson



Fast Point-Feature Label Placement for Dynamic Visualizations  

SciTech Connect

This paper presents a brand new approach for automated feature-point label de-confliction. It outlines a method for labeling the point-features on dynamic maps in real time without a pre-processing stage. The algorithm described provides an efficient, scalable, and exceptionally fast method of labeling interactive charts and diagrams, offering interaction speeds at multiple frames per second on maps with tens of thousands of nodes. To accomplish this, the algorithm employs an efficient approach -- called the "trellis strategy" -- along with a unique label candidate cost analysis, to determine the “least expensive” label configuration. The speed and scalability of this approach makes it suitable for the complex and ever-accelerating demands of interactive visual analytic applications.

Mote, Kevin D.



Quality criteria for the isotope dilution method with HRGC\\/MS  

Microsoft Academic Search

The isotope dilution method is characterized by the use of appropiately labelled standard compounds in conjunction with mass spectrometry coupled to chromatographic systems. Although the isotope dilution method seems to be a simple procedure, some quality criteria for sample preparation, measurement and quantification have to be considered. Furthermore, archiving and documentation of analysis data as well as the use of

B. Henkelmann; K.-W. Schramm; C. Klimm; A. Kettrup




Microsoft Academic Search

In animal studies it was necessary to detect in the exhaled air the O\\/; sub 2\\/ introduced in the blood stream in order to be able to recognize possible ; desiccation. Since radioactive oxygen isotopes with a half life long enough for ; the investigation do not exist, the stable oxygen isotope O¹⁸ was used for ; the labeling. The

G. Hubner; R. Ludewig; V. Gorisch; H. Birkenfeld



Dual Mode Fluorescent (18)F-PET Tracers: Efficient Modular Synthesis of Rhodamine-[cRGD]2-[(18)F]-Organotrifluoroborate, Rapid, and High Yielding One-Step (18)F-Labeling at High Specific Activity, and Correlated in Vivo PET Imaging and ex Vivo Fluorescence.  


The design of dual mode fluorescent-PET peptidic tracers that can be labeled with [(18)F]fluoride at high specific activity and high yield has been challenged by the short half-life of (18)F and its aqueous indolence toward nucleophilic displacement, that often necessitates multistep reactions that start with punctiliously dry conditions. Here we present a modular approach to constructing a fluorescent dimeric peptide with a pendant radioprosthesis that is labeled in water with [(18)F]fluoride ion in a single, user-friendly step. The modular approach starts with grafting a new zwitterionic organotrifluoroborate radioprosthesis onto a pentaerythritol core with three pendent alkynes that enable successive grafting of a bright fluorophore (rhodamine) followed by two peptides (cylcoRGD). The construct is labeled with [(18)F]fluoride via isotope exchange within 20 min in a single step at high specific activity (>3 Ci/?mol) and in good yield to provide 275 mCi and high radiochemical purity. Neither drying of the [(18)F]fluoride ion solution nor HPLC purification of the labeled tracer is required. Facile chemical synthesis of this dual mode tracer along with a user-friendly one-step radiolabeling method affords very high specific activity. In vivo PET images of the dual mode tracer are acquired at both high and low specific activities. At very high specific activity, i.e., 3.5 Ci/?mol, tumor uptake is relatively high (5.5%ID/g), yet the associated mass is below the limits of fluorescent detection. At low specific activity, i.e., 0.01 Ci/?mol, tumor uptake in the PET image is reduced by approximately 50% (2.9%ID/g), but the greater associated mass enables fluorescence detection in the tumor. These data highlight a facile production of a dual mode fluorescent-PET tracer which is validated with in vivo and ex vivo images. These data also define critical limitations for the use of dual mode tracers in small animals. PMID:25265337

Liu, Zhibo; Radtke, Mark Alex; Wong, May Q; Lin, Kuo-Shyan; Yapp, Donald T; Perrin, David M



Intra- and interspecific variability of carbon isotope composition (d13C) and water-use efficiency in 5 deciduous tree species growing a mixed stand in North-Eastern France  

NASA Astrophysics Data System (ADS)

Intra and interspecific variability in leaf gas exchange (net assimilation, stomatal conductance, transpiration, water use efficiency: WUE), in carbon isotope composition (d13C) and leaf characteristics related to photosynthesis was assessed in 5 trees species growing in a young broad-leaved mixed forest in North-Eastern France (Hesse, Lorraine). The studied species belong to contrasted functional groups of light tolerance: European beech (Fagus sylvatica) as shade tolerant species; sessile oak (Quercus petraea) and hornbeam (Carpinus betulus) as semitolerant species; silver birch (Betula pendula) and European aspen (Populus tremula) as shade intolerant species (pioneer species). Gas exchange was measured at leaf level in the upper and the lower canopy layers using a portable system (LI-6200, Licor). d13C signatures were determined in the sun and shade leaves in both bulk material and soluble sugars. Clear differences in bulk leaves and soluble sugars d13C and intrinsic WUEint were found among the investigated species, whatever the leaf location in the canopy. Within each tree species, shade leaves exhibited lower WUEint and more negative d13C than sun leaves. Little variability among trees was found for a given species. The 3 functional groups were separated by their leaf carbon content. Nevertheless each of the variables d13C, leaf mass area and nitrogen content, alone, could not separate the groups. A linear relationship was found between WUEint and d13C at the intraspecific level (r2 = 0.87 for leaves; r2 =0.89 for sugars) and at the interspecific level (r2 = 0.72 for bulk leaves). Nevertheless, this relationship differed from that of Farquhar et al. (1982), due to a different intercept, while the slope was the same. The causes of these variations are discussed. Key words: d13C; deciduous; mixed forest; WUEint; shade tolerance.

Zapater, M.; Breda, N.; Storchi, G.; Granier, A.



Isotope Tales: Remaining Problems, Unsolvable Questions, and Gentle Successes  

NASA Astrophysics Data System (ADS)

Earth's biomes function and adapt today as climate changes and ecosystems and the organisms within them adapt. Stable isotope biogeochemistry has had a major influence in understanding climate perturbations and continues to be an active area of research on many fronts. Banking on the success of compound specific stable isotope analyses of amino acids, nitrogen, carbon, and hydrogen isotopes continue to reveal subtle shifts in oceanic food webs and metabolic changes in microbes, plants, and animals. A biochemical understanding of exactly how organisms process and partition stable isotopes during metabolism remains unsolved, but is required if this field is to move beyond description to quantitation. Although the patterns of carbon and nitrogen isotopes are fairly well established in the common amino acids, we need to consider specifics: How do shifting metabolic pathways (metabolomics) influence the outcome of stable isotope partitioning? What influence does the gut microflora in animals have on isotopic labeling? What are the intramolecular isotope patterns of common amino acids and what do they tell us? What can be learned with other isotope systems, such as hydrogen? Results and ideas of how to move forward in this field will be presented starting at the molecular level and ending with ecosystems.

fogel, marilyn; bradley, christina; newsome, seth; filipp, fabian



Further development of IDGS: Isotope dilution gamma-ray spectrometry  

NASA Astrophysics Data System (ADS)

The isotope dilution gamma-ray spectrometry (IDGS) technique for determining the plutonium concentration and isotopic composition of highly radioactive spent-fuel dissolver solutions has been further developed. Both the sample preparation and the analysis have been improved. The plutonium isotopic analysis is based on high-resolution, low-energy gamma-ray spectrometry. The plutonium concentration in the dissolver solutions then is calculated from the measured isotopic differences among the spike, the dissolver solution, and the spiked dissolver solution. Plutonium concentrations and isotopic compositions of dissolver solutions analyzed from this study agree well with those obtained by traditional isotope dilution mass spectrometry (IDMS) and are consistent with the first IDGS experimental result. With the current detector efficiency, sample size, and a 100-min count time, the estimated precision is approximately 0.5 percent for Pu-239 and Pu-240 isotopic analyses and approximately 1 percent for the plutonium concentration analysis.

Li, T. K.; Parker, J. L.; Kuno, Y.; Sato, S.; Kamata, M.; Akiyama, T.


Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.  


Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon (13)C and nitrogen (15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8mg of the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix protein from 1L of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid. PMID:24662511

Kroupa, Tomáš; Prchal, Jan; Doležal, Michal; Ruml, Tomáš; Hrabal, Richard



Quantification of Pantothenic Acid and Folates by Stable Isotope Dilution Assays  

Microsoft Academic Search

Stable isotope dilution assays for the quantification of pantothenic acid and folates in foods by using four-fold labeled isotopomers of the vitamins as internal standards (IS) were developed. The use of labeled IS enabled to exactly correct losses during cleanup and derivatization.Pantothenic acid and its labeled isotopomer were detected as trimethylsilyl derivatives by gas chromatography–mass spectrometry. In starch a detection

Michael Rychlik; Achim Freisleben



ChemTeacher: Isotopes  

NSDL National Science Digital Library

ChemTeacher compiles background information, videos, articles, demonstrations, worksheets and activities for high school teachers to use in their classrooms. The Isotopes page includes resources for teaching students about the structure and uses of isotopes.



Carbon isotope discrimination of arctic and boreal biomes inferred from remote atmospheric measurements  

E-print Network

efficiency values that minimize the cost function covary. The optimal light use efficiency was 0.47 gC MJÃ?1 to Geographic Region: Arctic region; KEYWORDS: net primary production, light use efficiency, carbon isotopeCarbon isotope discrimination of arctic and boreal biomes inferred from remote atmospheric


Understanding Pesticide Labels  

E-print Network

, Division of Soil and Water Conservation. Understanding the Label Helps Reduce Environmental Problems Home) National Water Quality Initiative Funds and the Virginia Department of Conservation and Recreation along with the pests. Some pesticides used near water may contaminate the water supply. Continuous use

Liskiewicz, Maciej


Labeling and functionalizing amphipols for biological applications.  


Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes. PMID:24696186

Le Bon, Christel; Popot, Jean-Luc; Giusti, Fabrice



Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice  

SciTech Connect

During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-({sup 131}I) labeled intact antibodies or {sup 131}I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2.

Buchegger, F.; Pelegrin, A.; Delaloye, B.; Bischof-Delaloye, A.; Mach, J.P. (Univ. of Lausanne (Switzerland))



Stable isotope analysis of breath using the optogalvanic effect  

NASA Astrophysics Data System (ADS)

A new technique based on the optogalvanic effect has been developed for the measurement of stable isotope ratios in the carbon dioxide of exhaled breath. Data obtained before and after ingestion of harmless stable isotope labeled compounds, metabolized to carbon dioxide, can be used for sensitive noninvasive diagnostics of various disease conditions. The technique uses the specificity of laser resonance spectroscopy and achieves sensitivity and accuracy typical of sophisticated isotope ratio mass spectrometers. Using fixed frequency carbon dioxide lasers, 13C/12C ratios can be determined with a precision of 2 ppm with 100 second averaging times. Multiple samples can be analyzed simultaneously providing real time continuous calibration. In a first application, analysis of 13C/12C ratios in exhaled human breath after ingestion of 13C labeled urea is being developed as a diagnostic for the bacterium H-pylori, known to be the causative agent for most peptic and duodenal ulcers.

Murnick, Daniel E.; Colgan, M. J.; Lie, H. P.; Stoneback, D.



VoiceLabel: using speech to label mobile sensor data  

Microsoft Academic Search

Many mobile machine learning applications require collecting and labeling data, and a traditional GUI on a mobile device may not be an appropriate or viable method for this task. This paper presents an alternative approach to mobile labeling of sensor data called VoiceLabel. VoiceLabel consists of two components: (1) a speech-based data collection tool for mobile devices, and (2) a

Susumu Harada; Jonathan Lester; Kayur Patel; T. Scott Saponas; James Fogarty; James A. Landay; Jacob O. Wobbrock



Pesticide Labeling: Labeling Claims1 Frederick M. Fishel2  

E-print Network

PI-105 Pesticide Labeling: Labeling Claims1 Frederick M. Fishel2 1. This document is PI-105, one, professor, Agronomy Department, and Director, Pesticide Information Office; Florida Cooperative Extension pesticides safely. Read and follow directions on the manufacturer's label. The Institute of Food

Watson, Craig A.


Biosynthetic Approaches to Isotope Enrichment for Applications in Neutron Scattering and High Field NMR Spectroscopy: Methylotrophic  

SciTech Connect

Limitations in current isotopic labeling methods present a substantial bottleneck for the application of advanced structural techniques to many important biochemical problems. New tools are required to efficiently produce the necessary labeling patterns in biochemical precursors and incorporate them into protein molecules for structural studies. This project proposed involved one aspect of this problem, the development of expression vectors for a methylotrophic bacterium, Methylobacterium extorquens AM1. If high-level, efficient expression could be obtained in such a bacterium, it would be possible to use low-cost {sup 2}H- and/or {sup 13}C-labeled substrates such as methanol to label proteins. The Lidstrom laboratory at the University of Washington worked closely with the collaborators at Los Alamos National Laboratories in the development and use of these vectors. (1) Overexpression of a target gene, bacterial dehalogenase--This enzyme was expressed in Methylobacterium extorquens AM1 using a high level methanol-inducible promoter, the mxaF promoter. High expression was achieved, but most was in an insoluble form. They expressed this protein in a mutant lacking polybetahydroxybutyrate granules, and high expression was achieved, up to 10% of the total soluble protein. The recombinant protein was purified and shown to be active, with characteristics similar to the enzyme produced in E. coli. (2) Development of regulated expression systems--A number of regulated promoters were tested in M. extorquens AM1, the most promising of which appeared to be the E. coli lac promoter coupled to the Laciq regulator. The repressor was shown to be active and a chromosomal insertion construct was generated that repressed the low-level lac promoter activity in M. extorquens AM1. However, IPTG induced this system only poorly. A number of studies were carried out leading to the conclusion that IPTG entered the cell but was exported by one or more export pumps. Target genes for such pumps were mutated but none of these showed increased induction. A number of methods were used to permeabilize the cell, and a 2-fold increase in induction was obtained with one of these. The activity of the lac promoter was increased by inserting a recently-identified M. extorquens AM1 enhancer element upstream. The promoter increased in activity 5-6 fold with this addition. In summary, they have developed a suite of expression tools and host mutant strains for expressing a variety of heterologous proteins in this methylotroph. These are now available for testing by the LANL collaborators in labeling reactors to obtain labeled proteins of interest.

Mary E. lidstrom



Isotopically controlled semiconductors  

SciTech Connect

Semiconductor bulk crystals and multilayer structures with controlled isotopic composition have attracted much scientific and technical interest in the past few years. Isotopic composition affects a large number of physical properties, including phonon energies and lifetimes, bandgaps, the thermal conductivity and expansion coefficient and spin-related effects. Isotope superlattices are ideal media for self-diffusion studies. In combination with neutron transmutation doping, isotope control offers a novel approach to metal-insulator transition studies. Spintronics, quantum computing and nanoparticle science are emerging fields using isotope control.

Haller, Eugene E.



Labeling lake water with tritium  

USGS Publications Warehouse

A method of packaging tritiated water in a manner that facilitates safe handling in environmental labeling operations, and procedures followed in labeling a large body of water with a small volume of tritiated water are described. ?? 1963.

Frederick, B.J.



40 CFR 156.10 - Labeling requirements.  

Code of Federal Regulations, 2011 CFR

...ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS LABELING REQUIREMENTS FOR PESTICIDES AND DEVICES General Provisions § 156... —(1) Contents of the label. Every pesticide product shall bear a label...



Microgravity Science Glovebox - Labels  

NASA Technical Reports Server (NTRS)

Labels are overlaid on a photo (0003837) of the Microgravity Science Glovebox (MSG). The MSG is being developed by the European Space Agency (ESA) and NASA are developing the MSG for use aboard the International Space Station (ISS). Scientists will use the MSG to carry out multidisciplinary studies in combustion science, fluid physics and materials science. The MSG is managed by NASA's Marshall Space Flight Center (MSFC). Photo Credit: NASA/MSFC



Use of tritiated prostaglandins in metabolism studies. I: Evaluation of the kinetic isotope effect in the prostaglandin dehydrogenase reactions  

Microsoft Academic Search

Although numerous data exist concerning tritium kinetic isotope effect in enzymic reactions, little is related to the metabolism of tritiated prostaglandins. The present study reports an evaluation of the kinetic isotope effect which occurs during the oxidation of 15-hydroxyl group of tritium-labeled prostaglandins E2 and F2 alpha by the 15-hydroxyprostaglandin dehydrogenase and during the oxidation of 9-hydroxyl group of tritium-labeled

C. Moussard; D. Alber; C. Perruche; J. C. Henry



Nutrition Marketing on Food Labels  

ERIC Educational Resources Information Center

Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita



Identifier Labeling Using Graphical Models  

Microsoft Academic Search

In this paper, we apply Bayesian Networks to the labeling of arbitrary string identifiers from search results over a music database. We find that our models perform with a 58% labeling ac- curacy, with errors primarily occurring when la- beling string data not been seen during training. We also present a method for searching potential labelings which attempts to address

Brian Ferris; Stephen Friedman



E-print Network


Schaefer, Marcus


[Blood volume measurement of newborn using stable isotope 50Cr].  


A technique for the blood volume measurement of newborns was established in which nonradioactive 50Cr was used in patients for whom radioactive labels were not advisable. The red blood cells (RBC) in the newborn's blood withdrawn from umbilical cord after birth were tagged with enriched stable isotope 50Cr (96%, normal abundance 4.3%) and reinjected into the newborn. Blood samples (0.5 ml) were withdrawn at 30 min and thereafter at 6, 12, 24, 48, 72 and 120 hours old. Samples were centrifugalized and portion of RBC was then freeze-dried, weighed and sealed into polyethylene sheet bag together with 50Cr standard. Neutron irradiation was performed in the reactors of the JAERI with thermal neutron flux 5 X 10(13), 2 X 10(13), 8 X 10(13) cm-2s-1 at JRR-2, -3 and -4 respectively for 20 min and samples were left for about two weeks after irradiation. Induced radioactivity (51Cr, 59Fe) of the sample was measured with a Ge(Li) gamma-ray detector system and 4096 channels pulse height analyzer. Analysis of activity data was carried out by BOB-76 code. The RBC and total blood volume of the newborn was calculated using an isotopic dilution technique. We have investigated on tagging efficiency of 50Cr to RBC, washing effect and dilution rate by 50Cr content or 51Cr/59Fe ratio. Significant difference was observed in the total blood volume of newborns depending on the delivery style and in addition, it changed dynamically along the time elapsed after birth. PMID:4011960

Yamabayashi, H; Izumo, M; Motoki, R; Yamamoto, T; Nishida, H; Shin, S; Sato, K; Suzuki, Y



XAS molecular-level speciation and isotopes to assess the biogeochemistry of trace elements in soils and sediments : Few examples on Zn  

NASA Astrophysics Data System (ADS)

Since the mid of the 90's, multi-Collector Inductively-Coupled-Plasma Mass Spectrometry (MC-ICP-MS) offers the possibility of measuring the relative proportions of isotopes for a broad range of environmentally relevant elements labelled as ''non traditional isotopes'' (Cr, Fe, Ni, Cu, Zn, Se, Hg,...). These recent analytical possibilities have already been used in a large set of studies in Earth and Environmental Sciences to depict the biogeochemical behavior of these elements (Johnson et al., 2004; Weiss et al., 2008). However, establishing the link between the isotopes signature of a given element in a given system at a given time and its biogeochemical history (sources and pathways) is not always straightforward and complementary information on the actual speciation of this element can reveal very useful to address this issue. Since the end of the 80's, synchrotron-based X-Ray Absorption Spectroscopy has revealed as one of the most efficient way to directly assess the molecular-level speciation of an element in a natural system (Brown and Sturchio, 2002). Although this technique has been restricted to heavily polluted systems at the beginning because of its low detection limit, third- and fourth-generation synchrotron facilities now offer the possibility of obtaining this kind of information on very dilute systems, which makes it useful for almost all natural systems. The aim of this presentation will be to show how combining isotopes and molecular-level XAS speciation in natural and laboratory systems can reveal really powerful to help at understanding how isotopes fractionate in nature and, in turn, how these isotopes can be used to improve our understanding of the biogeochemical behavior of trace elements in continental systems (soils and sediments). Examples presented will concern Zn because this trace element is the most frequently encountered in soils and sediments and because it can act as a nutrient or as a poison, depending on its concentration.

Juillot, F.; Marechal-Chenevier, C.; Noël, V. S.; Morin, G.; Cacaly, S.; Louvat, P.; Telouk, P.; Hazemann, J.; Proux, O.; gelabert, A.; Jouvin, D.; Voegelin, A.; Mueller, B.; Brown, G. E.



Dual C, N labelling of terrestrial slugs (Deroceras reticulatum)  

Microsoft Academic Search

A simple, rapid and cost-effective laboratory method is described for labelling terrestrial slugs simultaneously with C and N. Slugs (Deroceras reticulatum) were provided with a mixture of [U-C6]glucose, N-enriched lettuce powder, and wheat bran. Assimilation efficiencies for C (24.2%) and N (27.4%) were not affected by feeding regimes offering ad libitum or restricted access to unlabelled food during the labelling

Jens Dyckmans; Olaf Schmidt



Regression-based label fusion for multi-atlas segmentation  

Microsoft Academic Search

Automatic segmentation using multi-atlas label fusion has been widely applied in medical image analysis. To simplify the label fusion problem, most methods implicitly make a strong assumption that the segmentation errors produced by different atlases are uncorrelated. We show that violating this assumption significantly reduces the efficiency of multi-atlas segmentation. To address this problem, we propose a regression-based approach for

Hongzhi Wang; Jung Wook Suh; Sandhitsu Das; John Pluta; Murat Altinay; Paul Yushkevich



Microfluidic Radiometal Labeling Systems for Biomolecules  

SciTech Connect

In a typical labeling procedure with radiometals, such as Cu-64 and Ga-68; a very large (~ 100-fold) excess of the non-radioactive reactant (precursor) is used to promote rapid and efficient incorporation of the radioisotope into the PET imaging agent. In order to achieve high specific activities, careful control of reaction conditions and extensive chromatographic purifications are required in order to separate the labeled compounds from the cold precursors. Here we propose a microfluidic approach to overcome these problems, and achieve high specific activities in a more convenient, semi-automated fashion and faster time frame. Microfluidic reactors, consisting of a network of micron-sized channels (typical dimensions in the range 10 â�� 300 �¼m), filters, separation columns, electrodes and reaction loops/chambers etched onto a solid substrate, are now emerging as an extremely useful technology for the intensification and miniaturization of chemical processes. The ability to manipulate, process and analyze reagent concentrations and reaction interfaces in both space and time within the channel network of a microreactor provides the fine level of reaction control that is desirable in PET radiochemistry practice. These factors can bring radiometal labeling, specifically the preparation of radio-labeled biomolecules such as antibodies, much closer to their theoretical maximum specific activities.

Reichert, D E; Kenis, P J. A.



Map labeling and its generalizations  

SciTech Connect

Map labeling is of fundamental importance in cartography and geographical information systems and is one of the areas targeted for research by the ACM Computational Geometry Impact Task Force. Previous work on map labeling has focused on the problem of placing maximal uniform, axis-aligned, disjoint rectangles on the plane so that each point feature to be labeled lies at the corner of one rectangle. Here, we consider a number of variants of the map labeling problem. We obtain three general types of results. First, we devise constant-factor polynomial-time-approximation algorithms for labeling point features by rectangular labels, where the feature may lie anywhere on the boundary of its label region and where labeling rectangles may be placed in any orientation. These results generalize to the case of elliptical labels. Secondly, we consider the problem of labeling a map consisting of disjoint rectilinear fine segments. We obtain constant-factor polynomial-time approximation algorithms for the general problem and an optimal algorithm for the special case where all segments are horizontal. Finally, we formulate a bicriteria version of the map-labeling problem and provide bicriteria polynomial- time approximation schemes for a number of such problems.

Doddi, S. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Computer Science]|[Los Alamos National Lab., NM (United States); Marathe, M.V. [Los Alamos National Lab., NM (United States); Mirzaian, A. [York Univ., Toronto, ON (Canada). Dept. of Computer Science; Moret, B.M.E. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Computer Science; Zhu, B. [City Univ. of Hong Kong (Hong Kong). Dept. of Computer Science]|[Los Alamos National Lab., NM (United States)




SciTech Connect

We study routing problems in time-dependent and edge and/or vertex-labeled transportation networks. Labels allow one to express a number of discrete properties of the edges and nodes. The main focus is a unified algorithm that efficiently solves a number of seemingly unrelated problems in transportation science. Experimental data gained from modeling practical situations suggest that the formalism allows interesting compromises between the conflicting goals of generality and efficiency. 1. We use edge/vertex labels in the framework of Formal Language Constrained Path Problems to handle discrete choice constraints. The label set is usually small and does not depend on the graph. Edge labels induct! path labels, which allows us to impose feasibility constraints on the set of paths considered as shortest path candidates. Second, we propose monotonic piecewise-linear traversal functions to represent the time-dependent aspect of link delays. The applications that can be modeled include scheduled transit and time-windows. 3. Third, we combine the above models and capture a variety of natural problems in transportatiou science such as time-window constrained trip-chaining. The results demonstrate the robustness of the proposed formalisms. As evidence for our claims of practical efficiency in a realistic setting, we report preliminary computational experience from TRANSIMS case studies of Portland, Oregon.

Barrett, C. L. (Christopher L.); Bisset, K. R. (Keith R.); Jacob, R. (Riko); Konjevod, G. (Goran); Marathe, M. V. (Madhav V.)



Tin isotope fractionation in terrestrial cassiterites  

NASA Astrophysics Data System (ADS)

The isotopic composition of tin has been measured in a range of cassiterites and pure reagents to assess the extent to which this element is isotopically fractionated in natural processes. Only two samples showed evidence of isotopic fractionation, and it is concluded that natural Sn isotope fractionation is small and uncommon. This feature reflects the world dominance of Sn-oxide ores over Sn-sulphide ores, and the highly efficient processes of Sn dissolution and precipitation which negate equilibrium and kinetic fractionation of Sn isotopes, respectively. The two samples which show slight fractionation are a highly purified metal and cassiterite from the Archaean Greenbushes pegmatite, Western Australia. The latter Sn is 0.15%o per mass unit heavier than our laboratory standard, whereas the former is 0.12%. per mass unit lighter. Although the cassiterite fractionation is considered to result from natural geological processes, the fractionation of purified Sn may be either natural or relate to the purification process. Isotopic fractionation of this magnitude has a negligible effect on the current best estimate of the atomic weight of Sn, but it does place a lower limit on its associated accuracy.

McNaughton, N. J.; Rosman, K. J. R.



16 CFR 309.17 - Labels.  

Code of Federal Regulations, 2010 CFR




Read the Label First! Protect Your Garden  


... environment if not used correctly. Read the Label First! Labels tell you: How to use a product ... PETS. FOLLOWTHESETIPSWHENSELECTING AND USING AGARDENPRODUCT. Read the Label First! Developed by the Consumer Labeling Initiative: A Government, ...


16 CFR 305.17 - Television labeling.  

Code of Federal Regulations, 2012 CFR

...or smaller. (ii) The manufacturer may include the ENERGY STAR logo on the label as illustrated in Sample Labels 10, 11...Agency covering the televisions to be labeled may add the ENERGY STAR logo to those labels. (g) Distribution of...



16 CFR 305.17 - Television labeling.  

Code of Federal Regulations, 2013 CFR

...or smaller. (ii) The manufacturer may include the ENERGY STAR logo on the label as illustrated in Sample Labels 10, 11...Agency covering the televisions to be labeled may add the ENERGY STAR logo to those labels. (g) Distribution of...



Oxazolinone derivative of leucine for GC-MS: a sensitive and robust method for stable isotope kinetic studies of lipoproteins  

Microsoft Academic Search

Stable isotope labeled amino acids are commonly used as endogenous tracers to study the metabolism of lipo- proteins. The determination of isotopic enrichment of par- ticular amino acids in apolipoproteins is carried out by gas chromatography mass spectrometry (GC-MS). This report describes a robust and sensitive derivative for analysis of d 3 - leucine by GC-MS and its utility in

Kevin P. Dwyer; P. Hugh; R. Barrett; Dick Chan; Jock I. Foo; Gerald F. Watts; Kevin D. Croft


Erythrocyte survival in children as studied by labeling with stable 50Cr.  


The survival of 50Cr- and 51Cr-labeled autologous and/or homologous erythrocytes was compared simultaneously in eight pediatric patients and one adult. 50Cr, a stable, nonradioactive nuclide, had values comparable to those of standard radioactive 51Cr labeling. The data also demonstrated the capability of 50Cr-51Cr labeling to reveal differences in survival between two populations of erythrocytes monitored simultaneously in the same individual. The technique permitted the use of the nonradioactive isotope in volumes of blood that are appropriate for the pediatric age group. PMID:984005

Glomski, C A; Pillay, K K; Macdougall, L G



Use of indium-111-labeled cells in measurement of cellular dynamics of experimental cardiac allograft rejection  

SciTech Connect

This study evaluates the kinetics and utility of infused indium-111-labeled cells in detecting rejection in ACI to Lewis rat heart allografts. Syngeneic leukocytes, lymph node lymphocytes, and platelets were isolated and labeled with indium-111 (/sup 111/In) oxine, respectively, and were infused i.v. into Lewis rats carrying beating ACI or syngeneic hearts from post-transplant days 0 to 6. Recipients were imaged serially at 24 hr after infusion of labeled cells followed by excision of both native and transplanted hearts for direct isotope count. Labeled leukocytes accumulative progressively in the allograft with the scan becoming positive by post-transplant day 4. The ratio of allograft to native heart isotope counts rose from 1.25 on day 1 to 10.07 (P less than 0.0001) on day 7. The Lewis recipients infused with labeled lymphocytes showed a positive scan on days 6 and 7 whereas the allograft to native heart isotope count ratio rose from 0.97 on day 1 to 5.33 (P less than 0.001) on day 7. Recipients infused with /sup 111/In-labeled platelets showed a positive scan on days 5 to 7 and the allograft to native heart isotope count ratio rose sharply from 2.56 on day 4 to 16.98 (P less than 0.005) on day 7. Syngeneic heart grafts failed to demonstrate significant accumulation of any of the labeled cell population. These studies confirm the importance of nonlymphocytic cells in cellular rejection, evaluate the kinetics of graft invasion by the various cell types, and suggest that the techniques used afford a method for a safe and an early detection of allograft rejection.

Oluwole, S.; Wang, T.; Fawwaz, R.; Satake, K.; Nowygrod, R.; Reemtsma, K.; Hardy, M.A.



Supplementing National Menu Labeling  

PubMed Central

The US Food and Drug Administration’s forthcoming national menu labeling regulations are designed to help curb the national obesity epidemic by requiring calorie counts on restaurants’ menus. However, posted calories can be easily ignored or misunderstood by consumers and fail to accurately describe the healthiness of foods. We propose supplemental models that include nutritional information (e.g., fat, salt, sugar) or specific guidance (e.g., “heart-healthy” graphics). The goal is to empower restaurant patrons with better data to make healthier choices, and ultimately to reduce obesity prevalence. PMID:23078494

White, Lexi C.



Hybrid isotope separation scheme  


A method of yielding selectively a desired enrichment in a specific isotope including the steps of inputting into a spinning chamber a gas from which a scavenger, radiating the gas with a wave length or frequency characteristic of the absorption of a particular isotope of the atomic or molecular gas, thereby inducing a photochemical reaction between the scavenger, and collecting the specific isotope-containing chemical by using a recombination surface or by a scooping apparatus.

Maya, Jakob (Brookline, MA)



Isotopes of Pennies  

NSDL National Science Digital Library

This lab activity from Science Netlinks is designed to explain the weighted averages that are used in average atomic mass calculations. Students can be expected to learn that isotopes of an element have different masses; that isotopes are atoms of the same element that have different numbers of neutrons; and that atomic mass is the weighted average of the naturally occurring isotopes of an element.

Netlinks, Science; Science, American A.


Isotope Ratio Mass Spectrometry.  


Isotope Ratio Mass Spectrometry (IRMS) is a specialized technique used to provide information about the geographic, chemical, and biological origins of substances. The ability to determine the source of an organic substance stems from the relative isotopic abundances of the elements which comprise the material. Because the isotope ratios of elements such as carbon, hydrogen, oxygen, sulfur, and nitrogen can become locally enriched or depleted through a variety of kinetic and thermodynamic factors, measurement of the isotope ratios can be used to differentiate between samples which otherwise share identical chemical compositions. Several sample introduction methods are now available for commercial isotope ratio mass spectrometers. Combustion is most commonly used for bulk isotopic analysis, whereas gas and liquid chromatography are predominately used for the real-time isotopic analysis of specific compounds within a mixture. Here, highlights of advances in instrumentation and applications within the last three years are provided to illustrate the impact of this rapidly growing area of research. Some prominent new applications include authenticating organic food produce, ascertaining whether or not African elephants are guilty of night-time raids on farmers' crops, and linking forensic drug and soil samples from a crime scene to a suspected point of origin. For the sake of brevity, we focus this Minireview on the isotope ratio measurements of lighter-elements common to organic sources; we do not cover the equally important field of inorganic isotope ratio mass spectrometry. PMID:19173039

Muccio, Zeland; Jackson, Glen P



Coral calcification: Use of radioactive isotopes and metabolic inhibitors to study the interactions with photosynthesis and respiration  

Microsoft Academic Search

In order to characterize the process of calcification in scleractinian corals, a series of laboratory experiments were conducted using radioactive isotopes. Labelled calcium, bicarbonate and glucose were used and the fates of the labelled tracers were followed in the skeleton and the tissue fractions of the coral Galaxea fascicularis. In addition, a variety of metabolic inhibitors were used to test

F. A. Al-Horani; S. A. Al-Rousan; R. S. Manasrah; M. Y. Rasheed



Estimating field metabolic rates of pinnipeds: doubly labelled water gets the seal of approval  

Microsoft Academic Search

Summary 1. Measures of the field metabolic rate of marine mammals are extremely difficult to make but they are essential for assessing the impacts of marine mammals on prey populations, and for assessing dive performance in relation to aerobic limits. 2. The doubly labelled water (DLW) method is an isotope-based technique for the estimation of the CO 2 production, and

C. E. Sparling; D. Thompson; M. A. Fedak; S. L. Gallon; J. R. Speakman



Label Scrambling During CID of Covalently Labeled Peptide Ions  

NASA Astrophysics Data System (ADS)

Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work, we demonstrate that in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low charge-state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification.

Borotto, Nicholas B.; Degraan-Weber, Nicholas; Zhou, Yuping; Vachet, Richard W.



Iridium-catalyzed H/D exchange: ligand complexes with improved efficiency and scope.  


Hydrogen isotope exchange (HIE) is one of the most attractive tools for the introduction of deuterium or tritium to an organic compound. Herein, iridium complexes with N,P-ligands, highly active catalysts for asymmetric double bond reductions, have been tested for their HIE capabilities. Electron-rich ligands, containing dicyclohexylphosphines or phosphinites, have been identified as excellent ligands for efficient deuterium incorporation. Substrates with strong directing groups, that is, pyridines, ketones, and amides, as well as weak ligating units, such as, nitro, sulfones, and sulfonamides, could be labeled efficiently. With the addition of tris(pentafluorophenyl) borane to the reaction mixture, also highly deactivating nitrile substituents were well tolerated in the reaction. Based on the excellent results obtained with the chiral ThrePhox ligand, a structurally simpler, achiral ligand was developed. The iridium complex containing this ligand, proved to be a powerful catalyst for HIE reactions. PMID:25043104

Parmentier, Michael; Hartung, Thomas; Pfaltz, Andreas; Muri, Dieter



Discovery of the Arsenic Isotopes  

E-print Network

Twenty-nine arsenic isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

Shore, A; Heim, M; Schuh, A; Thoennessen, M



Discovery of the Arsenic Isotopes  

E-print Network

Twenty-nine arsenic isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Shore; A. Fritsch; M. Heim; A. Schuh; M. Thoennessen



Discovery of the cadmium isotopes  

SciTech Connect

Thirty-seven cadmium isotopes have been observed so far and the discovery of these isotopes is discussed here. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

Amos, S. [National Superconducting Cyclotron Laboratory and Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48824 (United States); Thoennessen, M., E-mail: thoennessen@nscl.msu.ed [National Superconducting Cyclotron Laboratory and Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48824 (United States)



Discovery of the Cadmium Isotopes  

E-print Network

Thirty-seven cadmium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

Amos, S



Discovery of the Vanadium Isotopes  

E-print Network

Twenty-four vanadium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Shore; A. Fritsch; M. Heim; A. Schuh; M. Thoennessen



Discovery of the Tungsten Isotopes  

E-print Network

Thirty-five tungsten isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Fritsch; J. Q. Ginepro; M. Heim; A. Schuh; A. Shore; M. Thoennessen



Discovery of the Silver Isotopes  

E-print Network

Thirty-eight silver isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Schuh; A. Fritsch; J. Q. Ginepro; M. Heim; A. Shore; M. Thoennessen



Discovery of the Tin Isotopes  

E-print Network

Thirty-eight tin isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

S. Amos; M. Thoennessen



Discovery of the Indium Isotopes  

E-print Network

Thirty-eight indium isotopes (A = 98-135) have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

S. Amos; M. Thoennessen



Discovery of the Einsteinium Isotopes  

E-print Network

Seventeen einsteinium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Bury; A. Fritsch; J. Q. Ginepro; M. Heim; A. Schuh; A. Shore; M. Thoennessen



Discovery of the Mercury Isotopes  

E-print Network

Forty mercury isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

D. Meierfrankenfeld; M. Thoennessen



Discovery of the Cobalt Isotopes  

E-print Network

Twenty-six cobalt isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

T. Szymanski; M. Thoennessen



Discovery of the Cadmium Isotopes  

E-print Network

Thirty-seven cadmium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

S. Amos; M. Thoennessen



Discovery of the Calcium Isotopes  

E-print Network

Twenty four calcium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

J. L. Gross; M. Thoennessen



Discovery of the Titanium Isotopes  

E-print Network

Twentyfive titanium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

D. Meierfrankenfeld; M. Thoennessen



Discovery of the Iron Isotopes  

E-print Network

Twenty-eight iron isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Schuh; A. Fritsch; M. Heim; A. Shore; M. Thoennessen



Discovery of the Gold Isotopes  

E-print Network

Thirty-six gold isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Schuh; A. Fritsch; J. Q. Ginepro; M. Heim; A. Shore; M. Thoennessen



Discovery of the Barium Isotopes  

E-print Network

Thirty-eight barium isotopes have so far been observed; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

A. Shore; A. Fritsch; J. Q. Ginepro; M. Heim; A. Schuh; M. Thoennessen



Discovery of the Krypton Isotopes  

E-print Network

Thirty-two krypton isotopes have been observed so far; the discovery of these isotopes is discussed. For each isotope a brief summary of the first refereed publication, including the production and identification method, is presented.

M. Heim; A. Fritsch; A. Schuh; A. Shore; M. Thoennessen



In vivo gallbladder absorption: a new dual-isotope technique  

SciTech Connect

Available methods for measuring in vivo gallbladder absorption preclude the use of animals in which hepatic bile enters the gallbladder via accessory or aberrant channels. However, accessory bile ducts are present in many of the animal models currently used in gallstone research. The aim of this study, therefore, was to evaluate a new dual-isotope technique that corrects for accessory bile flow and to compare data on electrolyte and water absorption with those derived from the standard, single-isotope technique. Prairie dogs underwent gallbladder exclusion by cystic duct ligation and common bile duct cannulation. Carbon 14-polyethylene glycol-labeled lactated Ringer's solution was instilled into the gallbladder while tritiated cholic acid was administered intravenously to label the bile acid pool. There is no correlation between water or electrolyte absorption and time, nor between water and electrolyte absorption, when these parameters are calculated by the standard, single-isotope technique. In contrast, use of the dual-isotope technique quantifies accessory bile duct flow and yields a linear increase in water and electrolyte absorption, both of which are time dependent. These data suggest that the dual-isotope technique provides a means to accurately measure in vivo gallbladder absorption in animals with or without accessory bile ducts.

Conter, R.L.; Porter-Fink, V.; Denbesten, L.; Roslyn, J.J.



What's In A Food Label?  

NSDL National Science Digital Library

This article provides definitions, explanations and calculations that go into the Nutrition Facts labels mandated by federal law in 1990. These labels contain information such as serving size, ingredients, and a breakdown of calories. The article uses a sample food label and provides the conversions of calories per gram and shows pie charts of the components. Finally, there is a glossary of terms and their quantitative definitions.



Label Ranking Algorithms: A Survey  

NASA Astrophysics Data System (ADS)

Label ranking is a complex prediction task where the goal is to map instances to a total order over a finite set of predefined labels. An interesting aspect of this problem is that it subsumes several supervised learning problems, such as multiclass prediction, multilabel classification, and hierarchical classification. Unsurprisingly, there exists a plethora of label ranking algorithms in the literature due, in part, to this versatile nature of the problem. In this paper, we survey these algorithms.

Vembu, Shankar; Gärtner, Thomas


Gastric ulcer localization: Potential use of in vivo labeling  

SciTech Connect

A previous work suggests that sucralfate labeled by binding to Tc-99m HSA permits the visualization of gastric ulcers. Potential problems with this technique are: 1) decreased binding of sucralfate to ulcer sites due to the labeling method of binding to exogenous protein (HSA); 2) overlying activity that may obscure identification of the ulcer. Because of these problems we have examined the possibility of direct in vivo Tc-99m labeling of sucralfate after it has already bound to the ulcer. In vitro studies were done to determine the binding of Tc-99m pertechnetate to sucralfate in the presence of tin in HCl solution at pHs comparable to those found in the stomach. Rapid and efficient labeling was achieved with 75-95% of the label bound to sucralfate at 30 minutes. In vivo studies were performed in rabbits with aspirin induced ulcers and in ulcer free human volunteers. The animal studies confirm that orally administered Tc-99m pertechnetate will bind to previously ingested sucralfate and that the labeled material will bind to the ulcers. Tc-99m pertechnetate was also shown to bind well to previously ingested sucralfate in humans. The results suggest that it is possible to label sucralfate in vivo. This method would offer the following advantages: 1) a simpler labeling procedure; 2) the potential of increased sensitivity by delaying the labeling until much of the sucralfate not bound to ulcer has passed, and thus decreasing the activity that remains in the stomach; and also by leaving the protein binding sites of the sucralfate free to interact with the ulcer since no exogenous protein is involved in labeling.

Pera, A.; Rose, H.; Seavers, R.; Bekerman, C.; Pinsky, S.



Isotope - based Quantum Information  

E-print Network

This paper is brief review of three aspects of the isotope - based quantum information: computation, teleportation and cryptography. Our results demonstrate not only that entanglement exists in elementary excitation of isotope - mixed solids but also it can be used for quantum information processing.

Vladimir G. Plekhanov



(Carbon isotope fractionation inplants)  

SciTech Connect

The objectives of this research are: To develop a theoretical and experimental framework for understanding isotope fractionations in plants; and to develop methods for using this isotope fractionation for understanding the dynamics of CO{sub 2} fixation in plants. Progress is described.

O'Leary, M.H.




Microsoft Academic Search

The isotopic composition of sulfur was determined for a large number of ; specimens representing the important geochemicai phases in which sulfur is ; present. The most significant process that causes sulfur isotopic fractionation ; is the reduction of dissolved sulfate by bacteria, although other processes such ; as distillation of volcanic emanations, oxidation-reduction of HâS, SO\\/sub ; 2\\/, and

W. U. Ault; J. L. Kulp




Microsoft Academic Search

Sulfur isotope abundances of thirty-nine specimens of seventeen ; meteorites are reported. The results show again the remarkable constancy of the ; sulfur isotope ratios for meteorites of all types. However, differences in the ; Saâ\\/Sâ ratios of 0.4% would appear to be significant. The Smâ\\/; Sâ ratio for meteorites is discussed as a possible base level from which ;

H. G. Thode; H. B. Dunford



Labeled Cocaine Analogs  


Novel methods for positron emission tomography or single photon emission spectroscopy using tracer compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)napthyl Y in .beta. configuration is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, The compounds bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

Goodman, Mark M. (Atlanta, GA); Shi, Bing Zhi (Tucker, GA); Keil, Robert N. (Atlanta, GA)



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

PubMed Central

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

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



Pesticide Labeling: Miscellaneous Label Parts1 Frederick M. Fishel2  

E-print Network

PI-109 Pesticide Labeling: Miscellaneous Label Parts1 Frederick M. Fishel2 1. This document is PI. Fishel, professor, Agronomy Department, and Director, Pesticide Information Office; Florida Cooperative not signify our approval to the exclusion of other products of suitable composition. Use pesticides safely

Watson, Craig A.


Pesticide Labeling: Unique Product Labeling1 Frederick M. Fishel2  

E-print Network

PI-110 Pesticide Labeling: Unique Product Labeling1 Frederick M. Fishel2 1. This document is PI-110, professor, Agronomy Department, and Director, Pesticide Information Office; Florida Cooperative Extension not signify our approval to the exclusion of other products of suitable composition. Use pesticides safely

Watson, Craig A.


Adapter reagents for protein site specific dye labeling.  


Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 273-279, 2014. PMID:24599728

Thompson, Darren A; Evans, Eric G B; Kasza, Tomas; Millhauser, Glenn L; Dawson, Philip E



Leukocyte labeling with technetium-99m tin colloids.  


Triple density gradients of metrizamide in plasma (MP) were used to characterize label distribution in human leukocyte preparations incubated with 99mTc tin colloids. Less than 50% of the cell-associated radioactivity was specifically bound to leukocytes when heparinized blood was rotated with stannous fluoride colloid ([Tc]SFC). Labeling efficiency in leukocyte rich plasma (LRP) averaged 44%, of which greater than 90% was specifically bound to leukocytes. MP-gradient analysis also revealed that leukocyte labeling did not occur with stannous chloride colloid, nor when citrate was present during rotation with [Tc]SFC. When citrate was added after labeling to "solubilize" unbound [Tc]SFC, radiocolloid was removed from the leukocytes, indicating that the mechanism of [Tc]SFC labeling is adherence rather than phagocytosis. Technetium-labeled neutrophils exhibited normal in vitro chemotaxis and no lung uptake in vivo. Technetium-labeled mononuclear leukocytes, on the other hand, exhibited prolonged lung transit in vivo. Neither [Tc]SFC cell preparation showed signs of in vivo reoxidation to pertechnetate. PMID:3625299

Mock, B H; English, D



Problems and prospects in future applications of stable isotopes in the life sciences and medicine  

SciTech Connect

In the last decade, there has been a resurgence of interest in the use of stable isotopes of carbon, oxygen, and nitrogen in the life sciences and medicine fueled by the increased availability of the isotopes and isotopically labeled compounds and of instruments for their detection. Accelerated development of /sup 13/C, /sup 15/N, and /sup 17/ /sup 18/O can be expected in the future for studies of drug bioavailability, nutrition and body protein economy, viability of organs for transplant, and for non-invasive tests of metabolic diseases and dysfunctions. These accelerated developments depend on continued improvements in nmr and ms instrumentation and in methods for the synthesis of isotopically labeled compounds. The main part of this paper explores the possibilities of biosynthesis for the selective enrichment of natural products, especially amino acids, with /sup 13/C.

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



Electrochemical Isotope Effect and Lithium Isotope Separation Jay R. Black,  

E-print Network

Electrochemical Isotope Effect and Lithium Isotope Separation Jay R. Black, Grant Umeda, Bruce Dunn May 19, 2009; E-mail: The electrochemical separation of lithium isotopes is of growing interest due to the need for pure 6 Li and 7 Li isotopes in the nuclear industry.1 Here we present

Mcdonough, William F.


Intrinsic and Synthetic Stable Isotope Marking of Tsetse Flies  

PubMed Central

The sterile insect technique has been successfully used to eliminate tsetse populations in a number of programs. Program monitoring in the field relies on the ability to accurately differentiate released sterile insects from wild insects so that estimates can be made of the ratio of sterile males to wild males. Typically, released flies are marked with a dye, which is not always reliable. The difference in isotopic signatures between wild and factory-reared populations could be a reliable and intrinsic secondary marker to complement existing marking methods. Isotopic signatures are natural differences in stable isotope composition of organisms due to discrimination against the heavier isotopes during some biological processes. As the isotopic signature of an organism is mainly dependent on what it eats; by feeding factory-reared flies isotopically different diets to those of the wild population it is possible to intrinsically mark the flies. To test this approach unlabeled samples of Glossina pallidipes (Austen) (Diptera: Glossinidae) from a mass rearing facility and wild populations were analyzed to determine whether there were any natural differences in signatures that could be used as markers. In addition experiments were conducted in which the blood diet was supplemented with isotopically enriched compounds and the persistence of the marker in the offspring determined. There were distinct natural isotopic differences between factory reared and wild tsetse populations that could be reliably used as population markers. It was also possible to rear artificially isotopically labeled flies using simple technology and these flies were clearly distinguishable from wild populations with greater than 95% certainty after 85 days of “release”. These techniques could be readily adopted for use in SIT programs as complimentary marking techniques. PMID:21870965

Hood-Nowotny, Rebecca; Watzka, Margarete; Mayr, Leo; Mekonnen, Solomon; Kapitano, Berisha; Parker, Andrew



Direct immunofluorescent labeling of cells.  


In the direct immunofluorescent labeling technique, fluorochrome-labeled antibodies are used as probes for particular antigens or biomolecules. Cells, usually after appropriate fixation, are incubated with the antibodies to which fluorochromes have been directly conjugated. Following incubation, excess antibody is washed off with PBS and the cells are mounted on coverslips with antifade mounting medium. Immunofluorescent labeled cells are analyzed using a conventional fluorescence microscope or by confocal microscopy. Direct labeling has two major advantages: it requires only a single incubation with the labeled reagent, decreasing the number of steps in the staining procedure; and more importantly, provides minimal nonspecific staining and less background. Additionally, the direct labeling technique allows the use of two or more primary antibodies of the same species or isotype, avoiding the problems with secondary antibody staining. This method has multiple applications: to label simultaneously two or more antigens within the same cell or tissue sections; to characterize the subcellular distribution of biomolecules of interest, by concurrently labeling with antibodies to both the antigen of interest and to a known organelle; to investigate whether several antigens of interest are colocalized; and to phenotype cells, for which no specific markers are available, using an appropriate panel of antibodies. PMID:20012827

Pástor, Maria Veronica Dávila



Toolbox Safety Talk Chemical Labeling  

E-print Network

effective means of communicating chemical hazards. DEFINITIONS · Pictogram: a symbol plus other graphic in the workplace to be clearly labeled with the appropriate identification and warnings. A label may be affixed to to be taken to minimize or prevent adverse effects resulting from exposure to a hazardous chemical, improper

Pawlowski, Wojtek


Instant magnetic labeling of tumor cells by ultrasound in vitro  

NASA Astrophysics Data System (ADS)

Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 ?g/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling.

Mo, Runyang; Yang, Jian; Wu, Ed X.; Lin, Shuyu



Stable isotope enrichment using a plasma centrifuge  

NASA Astrophysics Data System (ADS)

A primary goal of the Department of Energy's Isotope Development and Production for Research and Applications Program (Isotope Program) within the Office of Nuclear Physics (NP) is to produce isotopes that are in short supply in the U.S. and of which there exists no or insufficient domestic commercial production capability. A vacuum arc plasma centrifuge is a rigid rotor column of metal plasma in which centrifugal forces re-distribute ions radially according to their mass/charge ratio. Early work demonstrated rotation at 2 million rpm and separation of various stable isotopes. The spinning plasma column had a Gaussian flux profile, peaked on the rigid rotor axis. This work adopts a more efficient approach, with the plasma created as a hollow column, wherein the flux is concentrated at larger radii where the centrifugal action is highest. By tailoring the vacuum arc discharge geometry, the rotation rate can also be increased to ˜10 million rpm. Data from Cu, Al and other metal plasmas will be presented and discussed in light of enriched stable isotopes needed for research and medicine.

Krishnan, Mahadevan; Bures, Brian; Madden, Robert



A simple method of labeling amyloid ? with quantum dots and ingestion of the labeled amyloid ? by astrocytes  

NASA Astrophysics Data System (ADS)

Steady labeling of amyloid beta (A?) is crucial for studying the ingestion and degradation of A? by astrocytes and unraveling a relevant regulation mechanism. Quantum dots (QDs) are an optimum labeling reagent for this because of their strong and steady fluorescence properties. In this paper, A? was labeled with QDs by a simple mixed incubation strategy, with a QD labeled A? complex (QDs-A?) being obtained. In the complex, QDs efficiently restrained the formation of ?-folding and fibrils of A?, while the graininess, dispersivity and fluorescence properties of the QDs hardly changed. The fluorescence microscopy imaging results showed that the astrocytes could ingest the QDs-A?. The QDs and A? did not separate from each other during the ingestion process, and the A? could be degraded subsequently.

Zhang, Jing; Jia, Xing; Qing, Hong; Xie, Hai-Yan



68 Ga-labeled multimeric RGD peptides for microPET imaging of integrin ? v ? 3 expression  

Microsoft Academic Search

Purpose  We and others have reported that 18F- and 64Cu-labeled arginine–glycine–aspartate (RGD) peptides allow positron emission tomography (PET) quantification of integrin ?v?3 expression in vivo. However, clinical translation of these radiotracers is partially hindered by the necessity of cyclotron\\u000a facility to produce the PET isotopes. Generator-based PET isotope 68Ga, with a half-life of 68 min and 89% positron emission, deserves special attention

Zi-Bo Li; Kai Chen; Xiaoyuan Chen



Off-label medication use.  


Prescribing medications for off-label uses is not illegal. Off-label prescribing includes using medications for unapproved indications; using a drug outside of the recommended dosage range or duration of use; using a drug in certain unapproved patient populations, such as those defined by age, sex, or particular clinical parameters; or intentionally using a medication in a patient who has a known contraindication. Medications would be considered appropriate for off-label use based on their known clinical pharmacology, evidence from clinical studies, and sometimes from the personal experience of the prescriber. The decision to use a drug off label should be based on a careful assessment of the patient's treatment history and the drug's potential risks and benefits. Patients should be given adequate informed consent about how the drug is being used off label and why, along with appropriate information about known risks and side effects. PMID:22897212

Howland, Robert H



Meteoritic Sulfur Isotopic Analysis  

NASA Technical Reports Server (NTRS)

Funds were requested to continue our program in meteoritic sulfur isotopic analysis. We have recently detected a potential nucleosynthetic sulfur isotopic anomaly. We will search for potential carriers. The documentation of bulk systematics and the possible relation to nebular chemistry and oxygen isotopes will be explored. Analytical techniques for delta(sup 33), delta(sup 34)S, delta(sup 36)S isotopic analysis were improved. Analysis of sub milligram samples is now possible. A possible relation between sulfur isotopes and oxygen was detected, with similar group systematics noted, particularly in the case of aubrites, ureilites and entstatite chondrites. A possible nucleosynthetic excess S-33 has been noted in bulk ureilites and an oldhamite separate from Norton County. High energy proton (approximately 1 GeV) bombardments of iron foils were done to experimentally determine S-33, S-36 spallogenic yields for quantitation of isotopic measurements in iron meteorites. Techniques for measurement of mineral separates were perfected and an analysis program initiated. The systematic behavior of bulk sulfur isotopes will continue to be explored.

Thiemens, Mark H.



Meteoritic Sulfur Isotopic Analysis  

NASA Astrophysics Data System (ADS)

Funds were requested to continue our program in meteoritic sulfur isotopic analysis. We have recently detected a potential nucleosynthetic sulfur isotopic anomaly. We will search for potential carriers. The documentation of bulk systematics and the possible relation to nebular chemistry and oxygen isotopes will be explored. Analytical techniques for delta33, delta34S, delta36S isotopic analysis were improved. Analysis ofmilligram samples is now possible. A possible relation between sulfur isotopes and oxygen was detected, with similar group systematics noted, particularly in the case of aubrites, ureilites and entstatite chondrites. A possible nucleosynthetic excess S-33 has been noted in bulk ureilites and an oldhamite separate from Norton County. High energy proton (approximately 1 GeV) bombardments of iron foils were done to experimentally determine S-33, S-36 spallogenic yields for quantitation of isotopic measurements in iron meteorites. Techniques for measurement of mineral separates were perfected and an analysis program initiated. The systematic behavior of bulk sulfur isotopes will continue to be explored.

Thiemens, Mark H.



Project EARTH-13-SHELLHCJ1: A combined isotopic and organic geochemical approach to reconstructing nutrient dynamics in the Mesozoic oceans*  

E-print Network

efficiency. This studentship will employ the novel approach of combining new and established isotope proxies, and an interest in exploring the uses of isotope and organic geochemistry to understand the past carbon cycleProject EARTH-13-SHELLHCJ1: A combined isotopic and organic geochemical approach to reconstructing

Henderson, Gideon


Sparse labeling of proteins: Structural characterization from long range constraints  

NASA Astrophysics Data System (ADS)

Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacte