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Improved folate extraction and tracing deconjugation efficiency by dual label isotope dilution assays in foods.  


A dual label stable isotope dilution assay was developed to trace the deconjugation efficiency of polyglutamic folate vitamers converted to their monoglutamic analogues. For this purpose, [(13)C(5)]-pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [(2)H(4)]-5-methyltetrahydrofolate, [(2)H(4)]-5-formyltetrahydrofolate, [(2)H(4)]-tetrahydrofolate, [(2)H(4)]-10-formylfolate, and [(2)H(4)]-folic acid. The [(2)H(4)]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [(13)C(5)]-pteroylheptaglutamate to the detection tracer [(13)C(5)]-folic acid, which was quantified along with unlabeled folic acid using [(2)H(4)]-folic acid as the internal standard. LC-MS/MS enabled the unequivocal differentiation of the three isotopologues. This tracing was used to optimize deconjugation efficiency, which was achieved by using 4-morpholineethanesulfonic acid buffer for extraction at pH 5.0 . The optimized assay revealed limits of detection for the folate vitamers ranging between 2.0 and 5.6 pmol per assay (equivalent to 2.2-6.6 ?g/100 g dry mass), recoveries ranging between 98 and 105% and relative standard deviations in inter-assay precision ranging between 2 and 6%. The assay was applied to quantitate folates in spinach, beans, cheeses, bread, wheat germs, and yeast . PMID:22235748

Mönch, Sabine; Rychlik, Michael



A novel design for a dual stable isotope continuous labeling chamber: results on labeling efficiency and C and N allocation in Andropogon gerardii  

NASA Astrophysics Data System (ADS)

The use of stable isotope enriched plant material can provide an unobstructed method of studying ecosystem nutrient dynamics between plants, soil, and atmosphere. However, the production of uniformly labeled perennial plant material is challenging due to plant physiological constraints and the mechanics of building and operating an isotope labeling system. In this study we present the design of a novel dual 13C and 15N continuous isotope labeling chamber located at Colorado State University. The chamber is equipped with automatic controls for CO2 concentration, temperature, and humidity, and has successfully been used to grow and label the tallgrass perennial Andropogon gerardii in pots from rhizomes. Three different nitrogen fertilization levels were applied to assess how substrate availability may alter growth and overall performance in the system. The efficiency of the 13C and 15N labeling chamber, its design and overall performance, as well as a full C, N, 13C, and 15N budget of the aboveground biomass, belowground biomass, and soil will be presented. Solid samples were analyzed on an EA-IRMS, while air samples from the chamber were analyzed using a precon-GC-IRMS system. The dual stable isotope labeled A. gerardii produced from this chamber will be used in a decomposition experiment to quantify the relative contribution of aboveground litter derived C to soil respiration, dissolved organic carbon, and various soil organic matter pools. Based on the results of our A. gerardii 13C and 15N labeling experiment we believe that this chamber design can be used to successfully produce dual stable isotope labeled plants for a wide variety of terrestrial nutrient flux experiments.

Soong, J.; Stewart, C.; Reuss, D.; Pinney, C.; Cotrufo, F. M.



Efficient synthesis of D-branched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase.  


D-Branched-chain amino acids (D-BCAAs) such as D-leucine, D-isoleucine, and D-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of D-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of D-BCAAs and production of D-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: D-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding D-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and D-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to D-leucine with an excellent yield (>99 %) and optical purity (>99 %). Using this system, we produced five different D-BCAAs labeled with stable isotopes: D-[1-(13)C,(15)N]leucine, D-[1-(13)C]leucine, D-[(15)N]leucine, D-[(15)N]isoleucine, and D-[(15)N]valine. The structure of each labeled D-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of D-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of D-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating D-BCAAs and their derivatives. PMID:23661083

Akita, Hironaga; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa



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.

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



Isotope-labeled immunoassays without radiation waste  

PubMed Central

The practice of immunoassay has experienced a widespread transition from radioisotopic labeling to nonisotopic labeling over the last two decades. Radioisotope labels have drawbacks that hamper their applications: (i) perceived radiation hazards of reagents, (ii) regulatory requirements and disposal problems of working with radioactive materials, and (iii) short shelf-life of the labeled reagents. The advantage of isotopic labeling is the incorporation into analytes without altering structure or reactivity, as is often the case with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay with the long-life isotope 14C as the label and accelerator mass spectrometer (AMS) as the detection system. AMS quantifies attomole levels of several isotopes, including 14C. With this exquisite sensitivity, the sensitivity of an immunoassay is limited by the Kd of the antibody and not the detection system. The detection limit of the assays for atrazine and 2,3,7,8-tetrachlorodibenzo-p-dioxin was 2.0 × 10?10 M and 2.0 × 10?11 M, respectively, approximately an order of magnitude below the standard enzyme immunoassay. Notably, <1 dpm (0.45 pCi) of 14C-labeled compound was used in each assay, which is well below the limit of disposal (50 nCi per g) as nonradioactive waste. Thus, endogenous reporter ligands quantified by AMS provide the advantages of an RIA without the associated problems of radioactive waste.

Shan, Guomin; Huang, Wei; Gee, Shirley J.; Buchholz, Bruce A.; Vogel, John S.; Hammock, Bruce D.



Simple, rapid method for the preparation of isotopically labeled formaldehyde  

SciTech Connect

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.

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



Multiple isotopic labels for quantitative mass spectrometry.  


Quantitative mass spectrometry is often performed using isotopically labeled samples. Although 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 nine deuteriums and a light form without deuterium). In the present report, two additional forms containing three and six deuteriums have been synthesized and tested. These additional isotopic tags perform identically to the previously reported tags; peptides labeled with the new TMAB reagents coelute from reversed-phase HPLC columns with peptides labeled with the lighter and heavier TMAB reagents. Altogether, these four 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



An Efficient Synthetic Strategy for Obtaining 4-Methoxy Carbon Isotope Labeled Combretastatin A-4 Phosphate and Other Z-Combretastatins1  

PubMed Central

Human cancer and other clinical trials under development employing combretastatin A-4 phosphate (1b, CA4P) should benefit from the availability of a [11C]-labeled derivative for position emission tomography (PET). In order to obtain a suitable precursor for addition of a [11C]methyl group at the penultimate step, several new synthetic pathways to CA4P were evaluated. Geometrical isomerization (Z to E) proved to be a challenge, but it was overcome by development of a new CA4P synthesis suitable for 4-methoxy isotope labeling.

Pettit, George R.; Minardi, Mathew D.; Hogan, Fiona; Price, Pat M.



Matching isotopic distributions from metabolically labeled samples  

PubMed Central

Motivation: In recent years stable isotopic labeling has become a standard approach for quantitative proteomic analyses. Among the many available isotopic labeling strategies, metabolic labeling is attractive for the excellent internal control it provides. However, analysis of data from metabolic labeling experiments can be complicated because the spacing between labeled and unlabeled forms of each peptide depends on its sequence, and is thus variable from analyte to analyte. As a result, one generally needs to know the sequence of a peptide to identify its matching isotopic distributions in an automated fashion. In some experimental situations it would be necessary or desirable to match pairs of labeled and unlabeled peaks from peptides of unknown sequence. This article addresses this largely overlooked problem in the analysis of quantitative mass spectrometry data by presenting an algorithm that not only identifies isotopic distributions within a mass spectrum, but also annotates matches between natural abundance light isotopic distributions and their metabolically labeled counterparts. This algorithm is designed in two stages: first we annotate the isotopic peaks using a modified version of the IDM algorithm described last year; then we use a probabilistic classifier that is supplemented by dynamic programming to find the metabolically labeled matched isotopic pairs. Such a method is needed for high-throughput quantitative proteomic metabolomic experiments measured via mass spectrometry. Results: The primary result of this article is that the dynamic programming approach performs well given perfect isotopic distribution annotations. Our algorithm achieves a true positive rate of 99% and a false positive rate of 1% using perfect isotopic distribution annotations. When the isotopic distributions are annotated given ‘expert’ selected peaks, the same algorithm gets a true positive rate of 77% and a false positive rate of 1%. Finally, when annotating using machine selected peaks, which may contain noise, the dynamic programming algorithm gives a true positive rate of 36% and a false positive rate of 1%. It is important to mention that these rates arise from the requirement of exact annotations of both the light and heavy isotopic distributions. In our evaluations, a match is considered ‘entirely incorrect’ if it is missing even one peak or containing an extraneous peak. If we only require that the ‘monoisotopic’ peaks exist within the two matched distributions, our algorithm obtains a positive rate of 45% and a false positive rate of 1% on the ‘machine’ selected data. Changes to the algorithm's scoring function and training example generation improves our ‘monoisotopic’ peak score true positive rate to 65% while obtaining a false positive rate of 2%. All results were obtained within 10-fold cross-validation of 41 mass spectra with a mass-to-charge range of 800–4000m/z. There are a total of 713 isotopic distributions and 255 matched isotopic pairs that are hand-annotated for this study. Availability: Programs are available via Contact:

McIlwain, Sean; Page, David; Huttlin, Edward L.; Sussman, Michael R.



Resonance Raman studies of isotopically labeled chloroperoxidase  

SciTech Connect

Chloroperoxidase (CPO) and cytochrome P450cam have been shown by several techniques to have similar active site properties. Recent resonance Raman investigations using isotopically enriched 34S-labeled samples have demonstrated thiolate ligation in the P450cam system. We report here on a number of parallel studies involving CPO. On the basis of isotopic labeling (34S, 13CO), we assign the Fe-S and Fe-CO stretching frequencies of CPO at 347 (-vFe-S) and 488 cm-1 (-vFe-CO). The differences of the -vFe-S and -vFe-CO in CPO and P450cam may suggest subtle differences in the thiolate binding in the two systems.

Bangcharoenpaurpong, O.; Champion, P.M.; Hall, K.S.; Hager, L.P.



Efficient Labeling of Collinear Sites  

Microsoft Academic Search

In this paper we study the map labeling problem where the sites to be labeled are restricted to a line L. Previous models studied in the map labeling literature fail to produce label placements (i.e. place each label next to the site it describes) without label overlaps for certain instances of the problem with dense point sets. To address this

Michael A. Bekos; Michael Kaufmann; Antonios Symvonis



Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels.  


A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method. PMID:9109351

Arlinghaus, H F; Kwoka, M N; Guo, X Q; Jacobson, K B



Performance of isobaric and isotopic labeling in quantitative plant proteomics.  


Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins. PMID:22452248

Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit; Campos, Francisco A P; Larsen, Martin R; Domont, Gilberto B; Roepstorff, Peter



Cyclic Enzymatic Solid Phase Synthesis of Isotopically Labeled DNA Oligonucleotides  

Microsoft Academic Search

Isotopic labeling of DNA using standard solid phase synthesis requires expensive phosphoramidites that are used in large excess. We have developed a protocol where enzymatic, cyclic, solid phase synthesis of DNA facilitates a more economical use of the less expensive labeled DNA triphosphates (dNTP). In this approach, the DNA template is immobilized on an epoxy-activated solid support. Both the support

Ahmed M. Khan; Subrata H. Mishra; Markus W. Germann



Synthesis of isotopically labelled SGLT inhibitors and their metabolites  

Microsoft Academic Search

Isotopically labelled analogues of two structurally very similar SGLT inhibitors AVE2268 (1a) and AVE8887 (1b) have been synthesized by various routes. The radioactive labelled [14C]-AVE2268 was prepared in five steps including a Friedel–Crafts acylation as the key step for the 14C-label introduction. For [14C]-AVE8887 the same synthetic approach was not successful and therefore an alternative thiophene metallation\\/Weinreb amide sequence was

Volker Derdau; Thorsten Fey; Jens Atzrodt



Intrinsic stable isotope labeling of plants for nutritional investigations in humans  

Microsoft Academic Search

Although plant foods provide an array of nutrients in the human diet, our knowledge of how efficiently these nutrients are absorbed has been limited by our ability to selectively monitor their absorption from a complex food matrix. Intrinsic labeling of plants with low-abundance stable isotopes can provide a safe, traceable product to investigate absorptive phenomena in the gut. Various techniques,

Michael A. Grusak



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.

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



Stable Isotope Labeling with Amino acids in Nematodes  

PubMed Central

We describe an approach for the accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted Stable Isotope Labeling with Amino acids in Cell culture (SILAC) for nematodes, by feeding worms a heavy lysine- and arginine-labeled E. coli strain. We also report a genetic solution to remove the arginine-to-proline conversion problem. Combining our approach with quantitative proteomics methods, we characterized the heatshock response in worms.

Larance, Mark; Bailly, Aymeric P.; Pourkarimi, Ehsan; Hay, Ronald T.; Buchanan, Grant; Coulthurst, Sarah; Xirodimas, Dimitris P.; Gartner, Anton; Lamond, Angus I.



Dimethyl isotope labeling assisted de novo peptide sequencing  

Microsoft Academic Search

Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl\\u000a labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions.\\u000a For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific\\u000a labeling of

Marco L. Hennrich; Shabaz Mohammed; A. F. Maarten Altelaar; Albert J. R. Heck



A rapid and robust method for selective isotope labeling of proteins  

PubMed Central

Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with 13C- and 15N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins.

Lin, Myat T.; Sperling, Lindsay J.; Frericks Schmidt, Heather L.; Tang, Ming; Samoilova, Rimma I.; Kumasaka, Takashi; Iwasaki, Toshio; Dikanov, Sergei A.; Rienstra, Chad M.; Gennis, Robert B.



Proteome Analysis using Selective Incorporation of Isotopically Labeled Amino Acids  

SciTech Connect

A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins were extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of using this method to unambiguously identify proteins isolated from E. coli.

Veenstra, Timothy D.; Martinovic, Suzana; Anderson, Gordon A.; Pasa-Tolic, Liljiana; Smith, Richard D.



Neutron capture radiography: a technique for isotopic labelling and analytical imaging with a few stable isotopes.  


NCR (neutron capture radiography) may be used successfully for the imaging of one of the stable isotopes of a few chemical elements (especially 6Li and 10B, possibly also 14N, 17O, and others) and for labelling experiments using these stable isotopes. Other physical techniques compete with NCR. However, NCR can remain extremely useful in a certain number of cases, because it is usually more easily done and is less expensive than the other techniques. PMID:16799738

Thellier, Michel; Ripoll, Camille



Automated Online Sequential Isotope Labeling for Protein Quantitation Applied to Proteasome Tissue-specific Diversity  

Microsoft Academic Search

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reduc- ing the accuracy of measurements. Here, we

Reinout Raijmakers; Celia R. Berkers; Annemieke de Jong; Huib Ovaa; Albert J. R. Heck; Shabaz Mohammed




Technology Transfer Automated Retrieval System (TEKTRAN)

To investigate the bioavailability of carotenoids from a green, leafy vegetable, isotopically-labeled kale was produced and used in a human feeding trial. Kale was chosen because of its high content of both beta-carotene and lutein. The kale was grown in a sealed chamber in the presence of 13C-ca...


Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance  

PubMed Central

Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the 3D structures under near physiological conditions, but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows specific segment(s) within a protein to be selectively examined by NMR thus significantly reducing the spectral complexity for large proteins and allowing a variety of solution-based NMR strategies to be applied. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50 kDa protein Csk (C-terminal Src Kinase) using expressed protein ligation methods.

Dongsheng, Liu; Xu, Rong; Cowburn, David



Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.  


Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-?-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard. PMID:22662968

Gómez-Cortés, Pilar; Brenna, J Thomas; Sacks, Gavin L



Isotopic Labeling of Red Cabbage Anthocyanins with Atmospheric 13-CO2  

Technology Transfer Automated Retrieval System (TEKTRAN)

Isotopic labeling of plants provides a unique opportunity for understanding metabolic processes. A significant challenge of isotopic labeling during plant growth is that isotopes must be administered without disrupting plant development and at sufficient levels for mass spectral analysis. We describ...


Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry.  


Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([2H3]-leucine, [13C6]arginine, and [2H4]lysine), [13C6]arginine proved to be the most suitable. Incorporation of [13C6]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [13C6]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling. PMID:16088002

Gruhler, Albrecht; Schulze, Waltraud X; Matthiesen, Rune; Mann, Matthias; Jensen, Ole N



Light speed labeling: efficient connected component labeling on RISC architectures  

Microsoft Academic Search

This article introduces two fast algorithms for connected component Labeling of binary images, a peculiar case of coloring.\\u000a The first one, Selkow\\u000a \\u000a DT\\u000a is pixel-based and a Selkow’s algorithm combined with the decision tree optimization technique. The second one called light speed labeling is segment-based line-relative labeling and was especially thought for commodity RISC architectures. An extensive benchmark on both

Lionel Lacassagne; Bertrand Zavidovique



Triple isotopic labeling and kinetic isotope effects: exposing H-transfer steps in enzymatic systems.  


Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644-2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues. PMID:21688781

Sen, Arundhuti; Yahashiri, Atsushi; Kohen, Amnon



Sequencing of Isotope-Labeled Small RNA Using Femtosecond Laser Ablation Time-of-Flight Mass Spectrometry  

NASA Astrophysics Data System (ADS)

A novel method for the analysis of sequences of small RNAs using nucleotide triphosphates labeled with stable isotopes has been developed using time-of-flight mass spectroscopy combined with femtosecond laser ablation (fsLA-TOF-MS). Small RNAs synthesized with nucleotides enriched in 13C and 15N were efficiently atomized and ionized by single-shot fsLA and the isotope ratios 13C/12C and 15N/14N were evaluated using the TOF-MS method. By comparing the isotope ratios among four different configurations, the number of nucleotide contents of the control RNA sample were successfully reproduced.

Kurata-Nishimura, Mizuki; Ando, Yoshinari; Kobayashi, Tohru; Matsuo, Yukari; Suzuki, Harukazu; Hayashizaki, Yoshihide; Kawai, Jun



Discovery of novel hippocampal neurogenic agents by using an in vivo stable isotope labeling technique.  


Neurogenesis occurs in discrete regions of adult mammalian brain, including the subgranular zone of the hippocampus. Hippocampal neurogenesis is enhanced by different classes of antidepressants, but screening for neurogenic actions of novel antidepressants has been inefficient because of limitations of 5-bromo-2'-deoxyuridine labeling techniques. We describe an efficient in vivo method for measuring hippocampal neurogenesis involving incorporation of the stable isotope, (2)H, into genomic DNA during labeling with (2)H(2)O (heavy water). Male rodents received 8 to 10% (2)H(2)O in drinking water; DNA was isolated from hippocampal progenitor cells or neurons. Label incorporation into progenitor cells of Swiss-Webster mice revealed subpopulation kinetics: 16% divided with t(1/2) of 2.7 weeks; the remainder did not divide over 1 year. Progenitor cell proliferation rates in mice were strain-dependent. Chronic antidepressant treatment for 3 weeks, with (2)H(2)O administered during the final week, increased progenitor cell proliferation across all the strains tested. Fluoxetine treatment increased (2)H incorporation into DNA of gradient-enriched neurons or flow-sorted neuronal nuclei 4 weeks after (2)H(2)O labeling, representing the survival and differentiation of newly divided cells into neurons. By screening 11 approved drugs for effects on progenitor cell proliferation, we detected previously unrecognized, dose-dependent enhancement of hippocampal progenitor cell proliferation by two statins and the anticonvulsant topiramate. We also confirmed stimulatory activity of other anticonvulsants and showed inhibition of progenitor cell proliferation by isotretinoin and prednisolone. In conclusion, stable isotope labeling is an efficient, high-throughput in vivo method for measuring hippocampal progenitor cell proliferation that can be used to screen for novel neurogenic drugs. PMID:16973885

Shankaran, Mahalakshmi; King, Chelsea; Lee, Jean; Busch, Robert; Wolff, Mary; Hellerstein, Marc K



Simplified synthesis of isotopically labeled 5,5-dimethyl-pyrroline N-oxide.  


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

Leinisch, Fabian; Jiang, Jinjie; Deterding, Leesa J; Mason, Ronald P



Hydrolysis of adenosine 5'-triphosphate: an isotope-labeling study  

SciTech Connect

A combination of /sup 18/O-labeling experiments and kinetic studies to clarify the nonenzymatic hydrolytic pathways of adenosine 5'-triphosphate (ATP) at pH values ranging from 0 to 8.3 has been used in this experiment. In 1 N and 0.1 N HCl, the data are consistent with the hypothesis that hydrolysis occurs by addition-elimination, with initial attack 93% ..gamma.. and 7% ..beta..; both lead only to ADP + P/sub i/. In the subsequent hydrolysis of the ADP to AMP + P/sub i/, attack is 83% ..beta.. and 17% ..cap alpha... At pH 8.3, the data are consistent with the hypothesis that hydrolsysis occurs by elimination-addition. Over the entire pH range studied, no oxygen exchange between water and ATP, ADP, or P/sub i/ was detected. Nonenzymatic hydrolysis and isotopic analysis of the resultant P/sub i/ comprise a preferred means of assaying the isotopic enrichment of (..gamma..-/sup 18/O)ATP to be used in studies of enzymatic processes.

Meyerson, S. (Standard Oil Co. (Indiana), Naperville, IL); Kuh, E.S.; Ramirez, F.; Marecek, J.F.



Carbon fluxes in natural plankton communities under elevated CO2 levels: a stable isotope labeling study  

NASA Astrophysics Data System (ADS)

The potential impact of rising carbon dioxide (CO2) on carbon fluxes in natural plankton communities was investigated during the 2005 PeECE III mesocosm study in Bergen, Norway. Triplicate mesocosms, in which a phytoplankton bloom was induced by nutrient addition, were incubated with 1×(~350 ?atm), 2×(~700 ?atm), and 3× present day CO2(~1050 ?atm) levels for 3 weeks. 13C labeled bicarbonate was added to all mesocosms to follow the transfer of carbon from dissolved inorganic carbon (DIC) into phytoplankton and subsequently heterotrophic bacteria, zooplankton, and settling particles. Isotope ratios of polar lipid fatty acids (PLFA) were used to infer the biomass and production of phytoplankton and bacteria. Phytoplankton PLFA were enriched within one day after label addition, while it took another 3 days before bacteria showed substantial enrichment. Group-specific primary production measurements revealed that coccolithophores grew faster than green algae and diatoms. Elevated CO2 had a significant positive effect on post-bloom biomass of green algae, diatoms, and bacteria. A simple model based on measured isotope ratios of phytoplankton and bacteria revealed that CO2 had no significant effect on the carbon transfer efficiency from phytoplankton to bacteria. There was no indication of enhanced settling based on isotope mixing models during the phytoplankton bloom. Our results suggest that CO2 effects are most pronounced in the post-bloom phase, under nutrient limitation.

de Kluijver, A.; Soetaert, K.; Schulz, K. G.; Riebesell, U.; Bellerby, R. G. J.; Middelburg, J. J.



MALDI Biotyper-Based Rapid Resistance Detection by Stable-Isotope Labeling.  


Against the background of increasing numbers of resistant microorganisms, the fast and cost-efficient detection of microbial resistance is an important clinical requirement for optimal therapeutic intervention. Current routine assays take at least 5 h, but in most cases an overnight incubation is necessary to identify resistant isolates. The usage of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiling in combination with growth media containing isotopically labeled amino acids facilitates the detection of resistant microorganisms after 3 h or less directly from the profile spectrum. Growing microorganisms incorporate isotopically labeled amino acids, increasing protein masses and thereby leading to mass shifts of their corresponding peaks in the profile spectra. In the presence of antibiotics, only resistant microorganisms are able to grow and to incorporate the labeled amino acids. This leads to a difference in the mass spectra of susceptible and resistant isolates, allowing their differentiation. In the presented study, we demonstrated the applicability of this novel approach for the detection of methicillin-resistant Staphylococcus aureus and tested different bioinformatics approaches for automated data interpretation. PMID:24006001

Sparbier, Katrin; Lange, Christoph; Jung, Jette; Wieser, Andreas; Schubert, Sören; Kostrzewa, Markus



Stable isotope labeling of mammals (SILAM) for in vivo quantitative proteomic analysis.  


Metabolic labeling of rodent proteins with ¹?N, a heavy stable isotope of nitrogen, provides an efficient way for relative quantitation of differentially expressed proteins. Here we describe a protocol for metabolic labeling of rats with an ¹?N-enriched spirulina diet. As a case study, we also demonstrate the application of ¹?N-enriched tissue as a common internal standard in quantitative analysis of differentially expressed proteins in neurodevelopment in rats at two different time points, postnatal day 1 and 45. We briefly discuss the bioinformatics tools, ProLucid and Census, which can easily be used in a sequential manner to identify and quantitate relative protein levels on a proteomic scale. PMID:23523555

Rauniyar, Navin; McClatchy, Daniel B; Yates, John R



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

SciTech Connect

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 all hydrogen isotopes mixed with the stable helium isotopes. The application of this mass spectrometer were demonstrated by analyzing mixtures of deuterated methanes. In addition, it was employed to study the radiochemical Witzbach exchange reaction between tritium and methanes. A laser-Raman spectrometer was designed for analysis of tritium-containing gases and was built from individual components. A tritium-compatible, metal-sealed Raman cuvette having windows with good optical properties and additional means for measuring the stray light was first used successfully in this work. The Raman spectra of the hydrogen isotopes were acquired in the pure rotation mode and in the rotation-vibration mode and were used for on. The deuterated methanes were measured by Raman spectroscopy, the wavenumbers determined were assigned to the corresponding vibrations, and the wavenumbers for the rotational fine-structure were summarized in tables. The fundamental Vibrations of the deuterated methanes produced Witzbach reactions were detected and assigned. The fundamental vibrations of the molecules were obtained with Raman spectroscopy for the first time in this work. The @-Raman spectrometer assembled is well suited for the analysis of tritium- containing gases and is practical in combination with mass spectrometry using an Omegatron, for studying gases used in fusion.

Jewett, J.R., Fluor Daniel Hanford



Labeling a Rectilinear Map More Efficiently  

Microsoft Academic Search

Given a rectilinear map consisting of n disjoint line segments, the correspondinglabeling problem is to place a rectangle at each segment, allowingthree possible positions, such that the rectangles do not intersect. Thisproblem has a decision and a height maximization version. This paperimproves results from Poon, Zhu and Chin: A polynomial time solutionfor labeling a rectilinear map (IPL 65 (1998), pp.

Tycho Strijk; Marc J. Van Kreveld



Uniform sup 13 C isotope labeling of proteins with sodium acetate for NMR studies: Application to human carbonic anhydrase II  

SciTech Connect

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of ({sup 13}C{sub 6}, 99%)glucose. The authors demonstrate here that uniformly (>95%) {sup 13}C and {sup 15}N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium (1,2-{sup 13}C{sub 2},99%)acetate as the sole carbon source and ({sup 15}N, 99%)ammonium chloride as the sole nitrogen source. In addition, the authors demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium (1-{sup 13}C, 99%)acetate and sodium (2-{sup 13}C, 99%)acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the {sup 1}H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium (1,2-{sup 13}C{sub 2}, 99%)acetate growth media is considerably less than the cost of the ({sup 13}C{sub 6}, 99%)glucose growth media. They report here the first published {sup 13}C and {sup 15}N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.

Venters, R.S.; Calderone, T.L.; Spicer, L.D.; Fierke, C.A. (Duke Univ., Durham, NC (USA))



A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)  

Microsoft Academic Search

Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural (“light”) amino acids are replaced by “heavy” SILAC amino acids. Cells grown in

Matthias Mann; Shao-En Ong



Energy-Efficient Appliance Labeling in China: Lessons for Successful Labeling Programs in Varied Markets.  

National Technical Information Service (NTIS)

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 supporti...



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


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 (13) C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of (13) C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the (13) C 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 (13) C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in (13) C-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 (13) C 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



Combination of online enzyme digestion with stable isotope labeling for high-throughput quantitative proteome analysis.  


Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10(5) Hela cells). PMID:22945397

Wang, Fangjun; Wei, Xiaoluan; Zhou, Hu; Liu, Jing; Figeys, Daniel; Zou, Hanfa



Highly enriched multiply-labeled stable isotopic compounds as atmospheric tracers  


Compounds multiply-labeled with stable isotopes and highly enriched in these isotopes are readily capable of detection in tracer experiments involving high dilutions. Thus, for example, /sup 13/C/sup 18/O/sub 2/ provides a useful tracer for following atmospheric pol lution produced as a result of fossil fuel burning. (Official Gazette)

Goldblatt, M.; McInteer, B.B.



Phytoplankton-bacteria coupling under elevated CO2 levels: a stable isotope labelling study  

NASA Astrophysics Data System (ADS)

The potential impact of rising carbon dioxide (CO2) on carbon transfer from phytoplankton to bacteria was investigated during the 2005 PeECE III mesocosm study in Bergen, Norway. Sets of mesocosms, in which a phytoplankton bloom was induced by nutrient addition, were incubated under 1× (~350 ?atm), 2× (~700 ?atm), and 3× present day CO2 (~1050 ?atm) initial seawater and sustained atmospheric CO2 levels for 3 weeks. 13C labelled bicarbonate was added to all mesocosms to follow the transfer of carbon from dissolved inorganic carbon (DIC) into phytoplankton and subsequently heterotrophic bacteria, and settling particles. Isotope ratios of polar-lipid-derived fatty acids (PLFA) were used to infer the biomass and production of phytoplankton and bacteria. Phytoplankton PLFA were enriched within one day after label addition, whilst it took another 3 days before bacteria showed substantial enrichment. Group-specific primary production measurements revealed that coccolithophores showed higher primary production than green algae and diatoms. Elevated CO2 had a significant positive effect on post-bloom biomass of green algae, diatoms, and bacteria. A simple model based on measured isotope ratios of phytoplankton and bacteria revealed that CO2 had no significant effect on the carbon transfer efficiency from phytoplankton to bacteria during the bloom. There was no indication of CO2 effects on enhanced settling based on isotope mixing models during the phytoplankton bloom, but this could not be determined in the post-bloom phase. Our results suggest that CO2 effects are most pronounced in the post-bloom phase, under nutrient limitation.

de Kluijver, A.; Soetaert, K.; Schulz, K. G.; Riebesell, U.; Bellerby, R. G. J.; Middelburg, J. J.



In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics.  


Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed. PMID:14730684

Ippel, Johannes H; Pouvreau, Laurice; Kroef, Toos; Gruppen, Harry; Versteeg, Geurt; van den Putten, Peter; Struik, Paul C; van Mierlo, Carlo P M



High-yield expression of isotopically labeled peptides for use in NMR studies  

PubMed Central

Fusion protein constructs of the 56 amino acid globular protein GB-1 with various peptide sequences, coupled with the incorporation of a histidine tag for affinity purification, have generated high-yield fusion protein constructs. Methionine residues were inserted into the constructs to generate pure peptides following CNBr cleavage, yielding a system that is efficient and cost effective for isotopic labeling of peptides for NMR studies and other disciplines such as mass spectroscopy. Six peptides of varying sequences and hydrophobicities were expressed using this GB-1 fusion protein technique and produced soluble fusion protein constructs in all cases. The ability to easily express and purify recombinant peptides in high yields is applicable for biomedical research and has medicinal and pharmaceutical applications.

Lindhout, Darrin A.; Thiessen, Angela; Schieve, Dean; Sykes, Brian D.



One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains.  


Amino acid selective isotope labeling is a useful approach to simplification of nuclear magnetic resonance (NMR) spectra of large proteins. Cell-free protein synthesis offers essentially unlimited flexibility of labeling patterns but is labor-intensive and expensive. In vivo labeling is simple in principle but generally requires auxotrophic strains, inhibitors of amino acid synthesis, or complex media formulations. We describe a simple procedure for amino acid selective labeling of proteins expressed in prototrophic Escherichia coli strains. Excellent labeling selectivity was achieved for histidine, lysine, methionine, and alanine. Simplicity and robustness of this protocol make it a useful tool for protein NMR. PMID:22538396

O'Grady, Christopher; Rempel, Benjamin L; Sokaribo, Akosiererem; Nokhrin, Sergiy; Dmitriev, Oleg Y



Convenient synthesis of stable deuterium-labeled alkylpyrazines for use in stable isotope dilution assays.  


Stable isotope dilution assays (SIDA) provide for accurate and precise quantitation of aroma components, such as alkylpyrazines, which are often present in low concentrations in complex food matrices. The unavailability of labeled standards is the main limitation to the widespread use of SIDA. This study describes the chlorination of several alkylpyrazines to form the corresponding chloroalkylpyrazine compounds, which are efficient starting materials for the synthesis of deuterium-labeled alkylpyrazines, namely [(2)H3]-2-methylpyrazine (d-1), [(2)H5]-2-ethylpyrazine (d-2), [(2)H3]-2,3(or 6)-dimethylpyrazine (d-3A, d-3B), [(2)H3]-2,[(2)H3]-6-dimethylpyrazine (d-3C), [(2)H5]-2,[(2)H5]-6-diethylpyrazine (d-4), [(2)H5]-2-ethyl-3(or 6)-methylpyrazine (d-5A, d-5B), 2,[(2)H3]-3,5-trimethylpyrazine (d-6), [(2)H5]-2-ethyl-3,6-dimethylpyrazine (d-7), [(2)H5]-2-ethyl-3,5-dimethylpyrazine (d-8), and 2,3-diethyl-[(2)H3]-5-methylpyrazine (d-9), which were obtained in good yields (57-100%) and high purities (86-98%). These stable isotopes were used as internal standards in SIDA to accurately and precisely determine selected alkylpyrazines in commercial peanut butter, cocoa powder, and instant coffee. 2,3-Diethyl-5-methylpyrazine (p-9) and 2-ethyl-3,5-dimethylpyrazine (p-8), despite their low abundance, had the highest odor-active values among the 13 pyrazines quantified in all products due to their very low odor thresholds. PMID:23528050

Fang, Mingchih; Cadwallader, Keith R



Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis.  


Nonenzymatic asparagine (Asn) deamidation is one of the commonly observed posttranslational modifications of proteins. Recent development of several specific analytical methods has allowed for efficient identification and differentiation of the deamidation products (i.e., isoaspartate [isoAsp] and aspartate [Asp]). Isotope labeling of isoAsp and Asp that are generated during sample preparation by 18O has been developed and can differentiate isoAsp and Asp as analytical artifacts from those present in the samples prior to sample preparation for an accurate quantitation. However, the 18O labeling procedure has a limitation due to the additional incorporation of up to two 18O atoms into the peptide C-terminal carboxyl groups. Variability in the incorporation of 18O atoms into the peptide C-terminal carboxyl groups results in complicated mass spectra and hinders data interpretation. This limitation can be overcome by the dissection of the complicated mass spectra using a calculation method presented in the current study. The multiple-step calculation procedure has been successfully employed to determine the levels of isoAsp and Asp that are present in the sample prior to sample treatment. PMID:23017877

Liu, Hongcheng; Wang, Fengqiang; Xu, Wei; May, Kimberly; Richardson, Daisy



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



Plasma appearance of labeled  -carotene, lutein, and retinol in humans after consumption of isotopically labeled kale  

Microsoft Academic Search

The bioavailability of carotenoids from kale was investigated by labeling nutrients in kale with 13 C, feeding the kale to seven adult volunteers, and analyzing serial plasma samples for labeled lutein, ? -carotene, and retinol. Ingested doses of labeled carotenoids were 34 ? mol for ? -carotene and 33 ? mol for lutein. Peak plasma concentra- tions, areas under the

Janet A. Novotny; Anne C. Kurilich; Steven J. Britz; Beverly A. Clevidence



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



Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip.  


Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO(2)) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible. PMID:22975804

Polat, Ayse Nur; Kraiczek, Karsten; Heck, Albert J R; Raijmakers, Reinout; Mohammed, Shabaz



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)



Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA.  

PubMed Central

A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR. Images

Batey, R T; Inada, M; Kujawinski, E; Puglisi, J D; Williamson, J R



Stable isotope labeling of oligosaccharide cell surface antigens  

SciTech Connect

The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

Unkefer, C.J.; Silks, L.A. III; Martinez, R.A. [and others



Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics  

Microsoft Academic Search

Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and\\/or arginine), one of them

Tamar Geiger; Jacek R Wisniewski; Juergen Cox; Sara Zanivan; Marcus Kruger; Yasushi Ishihama; Matthias Mann



Efficient excitation of media for laser isotope separation  

Microsoft Academic Search

A theoretical investigation is made of the photoionisation of a three-level medium intended for laser isotope separation. The results show that the highest ionisation efficiency can be achieved by dividing the process into two stages: coherent population inversion and photoionisation. A study is made of the possibility of inversion of three-level systems for various detunings of the laser frequencies from

S. K. Borisov; M A Kuzmina; V. A. Mishin



An air-tolerant approach to the carbonylative suzuki-miyaura coupling: applications in isotope labeling.  


Carbonylative Suzuki-Miyaura coupling conditions have been developed that proceed without the exclusion of oxygen and in the presence of nondegassed and nondried solvents. By adapting the method to a two-chamber setup, the direct handling of carbon monoxide, produced from stable CO precursors, is avoided. The protocol afforded the desired benzophenones with excellent functional group tolerance and in good yields. Substituting the CO precursor, in the CO-producing chamber, with its carbon-13 labeled version generated the corresponding carbon-13 labeled benzophenones. Finally, the developed system was applied in the synthesis and isotope labeling of two pharmaceuticals, nordazepam and Tricor. PMID:24004340

Ahlburg, Andreas; Lindhardt, Anders T; Taaning, Rolf H; Modvig, Amalie E; Skrydstrup, Troels



Labeling of Human Serum Albumin with Stable Isotope of Bromine; an in Vitro Study  

Microsoft Academic Search

Background: Possibility to trace-label albumin with isotopes results in information concerning its synthesis, breakdown, and distribution in the intra and extra cellular spaces. The iodination of albumin is a widespread procedure used in scientific studies. Bromine not only is more reactive and less expensive than io- dine, but bonds more easily with many elements. Therefore, it could be a suitable

D. Amanat



Quantitative approaches for analysing fluxes through plant metabolic networks using NMR and stable isotope labelling  

Microsoft Academic Search

The quantitative analysis of metabolic networks is a prerequisite for understanding the integration and regulation of plant metabolism and for devising rational approaches for manipulating resource allocation in plants. The analysis of steady state stable isotope labelling experiments using nuclear magnetic resonance (NMR) spectroscopy has developed into a powerful method for determining these fluxes in micro-organisms and its application to

N. J. Kruger; R. G. Ratcliffe; A. Roscher



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

Microsoft Academic Search

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

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



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

Microsoft Academic Search

We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose

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



Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways  

PubMed Central

Background Metabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems. Methodology/Principal Findings The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 1H and 13C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each 13C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient 13C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts. Conclusions/Significance Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of 13C atoms of given metabolites on development-dependent changes in the 56 identified 13C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism.

Chikayama, Eisuke; Suto, Michitaka; Nishihara, Takashi; Shinozaki, Kazuo; Hirayama, Takashi; Kikuchi, Jun




Microsoft Academic Search

The preparation of isotopically labeled pharma ceutical and diagnostic agents has seen an almost exponential growth in recent years. The majority of labeled materials available are inorganic compounds or high-molecular-weight organic substances con taiiing a chemisorbed or otherwise bound radioac tive isotope. Essentially all of the commercially available isotopes have half-lives of 6 hr or more. It is our intention

D. R. Christman; P. Wolf


Efficient methods and practical guidelines for simulating isotope effects  

NASA Astrophysics Data System (ADS)

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 16O/18O 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.

Ceriotti, Michele; Markland, Thomas E.



Global Potential of Energy Efficiency Standards and Labeling Programs  

SciTech Connect

This report estimates the global potential reductions in greenhouse gas emissions by 2030 for energy efficiency improvements associated with equipment (appliances, lighting, and HVAC) in buildings by means of energy efficiency standards and labels (EES&L). A consensus has emerged among the world's scientists and many corporate and political leaders regarding the need to address the threat of climate change through emissions mitigation and adaptation. A further consensus has emerged that a central component of these strategies must be focused around energy, which is the primary generator of greenhouse gas emissions. Two important questions result from this consensus: 'what kinds of policies encourage the appropriate transformation to energy efficiency' and 'how much impact can these policies have'? This report aims to contribute to the dialogue surrounding these issues by considering the potential impacts of a single policy type, applied on a global scale. The policy addressed in this report is Energy Efficient Standards and Labeling (EES&L) for energy-consuming equipment, which has now been implemented in over 60 countries. Mandatory energy performance standards are important because they contribute positively to a nation's economy and provide relative certainty about the outcome (both timing and magnitudes). Labels also contribute positively to a nation's economy and importantly increase the awareness of the energy-consuming public. Other policies not analyzed here (utility incentives, tax credits) are complimentary to standards and labels and also contribute in significant ways to reducing greenhouse gas emissions. We believe the analysis reported here to be the first systematic attempt to evaluate the potential of savings from EES&L for all countries and for such a large set of products. The goal of the analysis is to provide an assessment that is sufficiently well-quantified and accurate to allow comparison and integration with other strategies under consideration.

McNeil, Michael A; McNeil, Michael A.; Letschert, Virginie; de la Rue du Can, Stephane



Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme.  


Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ?-amine of a specific lysine on the other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals. PMID:21962295

Castañeda, Carlos; Liu, Jia; Chaturvedi, Apurva; Nowicka, Urszula; Cropp, T Ashton; Fushman, David



Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme  

PubMed Central

Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This however has been a major impediment in the ubiquitin field. Here we present a robust, efficient, and widely accessible method for controlled iterative non-enzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ?-amine of a specific lysine on the other ubiquitin. We augment the non-enzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked di-ubiquitin, and a mixed-linkage Lys33,Lys11-linked tri-ubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.

Castaneda, Carlos; Liu, Jia; Chaturvedi, Apurva; Nowicka, Urszula; Cropp, T. Ashton; Fushman, David



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


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 R(1rho) relaxation dispersion spectroscopy to explore exchange processes on the microsecond to millisecond time scale, 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



Regression analysis for comparing protein samples with 16O\\/18O stable-isotope labeled mass spectrometry  

Microsoft Academic Search

Motivation: Using stable isotopes in global proteome scans, labeled molecules from one sample are pooled with unlabeled molecules from anothersampleandsubsequentlysubjectedtomass-spectralanalysis. Stable-isotope methodologies make use of the fact that identical molecules of different stable-isotope compositions are differentiated in a mass spectrometer and are represented in a mass spectrum as distinct isotopic clusters with a known mass shift. We describe two multivariable

J. E. Ecker-passow; Ann L. Oberg; Terry M. Therneau; C. J. Mason; Douglas W. Mahoney; K. L. Johnson; J. E. Olson; H. R. Bergen III




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



Tests of isotopic separation efficiency of palladium packed columns  

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 presence were tested using an isotope displacement method. A simple computer model was also developed to calculate the number of theoretical separation stages based on the test results. The effects of column diameter, metal foam presence and gas flow rate were identified. (authors)

Heung, L. K.; Staack, G. C.; Klein, J. E.; Jacobs, W. D. [Savannah River National Laboratory, 773-A, Savannah River Site, Aiken, SC 29808 (United States)



Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay.  


We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ?35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR. PMID:23916531

Bhate, Manasi P; Wylie, Benjamin J; Thompson, Ameer; Tian, Lin; Nimigean, Crina; McDermott, Ann E



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.

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



Trypsin is the Primary Mechanism by which the 18O Isotopic Label is Lost in Quantitative Proteomic Studies  

PubMed Central

Labeling with 18O is currently one of the most commonly used methods for incorporating a stable isotopic label into samples for comparative proteomic studies. In this approach, isotopic labeling involves the enzymatic digestion, typically performed with trypsin, of a protein population in 18O water, which incorporates the stable isotope into the C-termini of the newly formed peptides. Although trypsin is often used to facilitate isotopic incorporation after digestion, it is typically overlooked that this same mechanism can lead to isotopic loss even under conditions such as low pH where it is assumed that trypsin is inactive. To examine the role trypsin plays in isotopic loss, several experiments were performed on the rate of de-labeling under conditions relevant to multidimensional proteomic experiments. Results from these studies demonstrate that enzyme facilitated exchange of 18O in the peptide with 16O in the aqueous solvent was the major process by which the label is removed from the peptides, even under conditions of low pH and temperature where trypsin is thought to be inactive. This study brings the rapid, tryptic facilitated exchange to the attention of laboratories using this scheme in order to prevent inaccuracies in quantitative labeling due to loss of the isotopic label.

Angel, Peggi M.; Orlando, Ron



Determining metal assimilation efficiency in aquatic invertebrates using enriched stable metal isotope tracers  

USGS Publications Warehouse

We employ a novel approach that combines pulse-chase feeding and multi-labelled stable isotopes to determine gut passage time (GPT), gut retention time (GRT), food ingestion rate (IR) and assimilation efficiency (AE) of three trace elements for a freshwater gastropod. Lettuce isotopically enriched in 53Cr, 65Cu and 106Cd was fed for 2 h to Lymnaea stagnalis. The release of tracers in feces and water was monitored for 48 h, during which unlabelled lettuce was provided ad libidum. The first defecation of 53Cr occurred after 5 h of depuration (GPT), whereas 90% of the ingested 53Cr was recovered in the feces after 22.5 h of depuration (GRT). 53Chromium was not significantly accumulated in the soft tissues upon exposure. In contrast, 65Cu and 106Cd assimilation was detectable for most experimental snails, i.e., 65/63Cu and 106/114Cd ratios in exposed snails were higher than those for controls. Food IR during the labelled feeding phase was 0.16 ?? 0.07 g g-1 d-1. IR was inferred from the amount of 53Cr egested in the feces during depuration and the concentration of 53Cr in the labelled lettuce. Assimilation efficiencies (??95% CI) determined using mass balance calculations were 84 ?? 4% for Cu and 85 ?? 3% for Cd. The ratio method yields similar AE estimates. Expanding the application of this novel stable isotope tracer technique to other metals in a wide variety of species will provide unique opportunities to evaluate the interplay between digestive processes and dietary influx of metals. Understanding the biological processes that modulate dietborne metal uptake is crucial to assess the toxicity of dietborne metals. ?? 2007 Elsevier B.V. All rights reserved.

Croteau, M. -N.; Luoma, S. N.; Pellet, B.




Technology Transfer Automated Retrieval System (TEKTRAN)

The purposes of this study were to quantitate the impact of the duration of infusion and choice of stable isotope of glucose on measures of glucose rate of appearance (glucose Ra) and to determine whether the differences observed were due to tracer recycling via the glycogen pool (direct pathway) or...


Subnivean decomposition in a Colorado subalpine forest - insights from an isotope labeling experiment  

NASA Astrophysics Data System (ADS)

A growing body of evidence has highlighted the importance of winter ecological processes for carbon cycling in many temperate regions, including subalpine forests of the western United States. We conducted an experiment to examine decomposition dynamics of ponderosa pine needle litter in a high-elevation subalpine forest in the Rocky Mountains of Colorado. Pine saplings were isotopically-labeled in a laboratory growth chamber, providing a litter source that was highly enriched in carbon-13 (+2500 permil). Enriched pine needles were applied to the forest floor of the Niwot Ridge AmeriFlux site in late fall. Decomposition of the labeled litter was monitored over one winter (as CO2) using tunable diode laser absorption spectroscopy in the field. Use of this technology allowed high-resolution examination of the fluxes of labeled subnivean respiration. The following summer, soils were collected and 52% of the C label remained, primarily in the organic soil horizon. Phospholipid fatty acid (PLFA) isotope analysis indicated that the majority of the remaining litter C was associated with fungi, but some was recovered in gram positive and negative bacterial groups. The dominant fungi that grew on the labeled needles were identified by 18S ribosomal DNA sequencing. Fungal phyla growing on the needles included Zygomycota, Ascomycota and Basidiomycota, some of which were known from our previous work, but several were not closely related to previously described snow molds.

Bowling, D. R.; Bird, J. A.; Schmidt, S. K.



Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution  

PubMed Central

The unique biology of a neoplasm is reflected by its distinct molecular profile compared with normal tissue. To understand tumor development better, we have undertaken a quantitative proteomic search for abnormally expressed proteins in colonic tumors from ApcMin/+ (Min) mice. By raising pairs of Min and wild-type mice on diets derived from natural-abundance or 15N-labeled algae, we used metabolic labeling to compare protein levels in colonic tumor versus normal tissue. Because metabolic labeling allows internal control throughout sample preparation and analysis, technical error is minimized as compared with in vitro labeling. Several proteins displayed altered expression, and a subset was validated via stable isotopic dilution using synthetic peptide standards. We also compared gene and protein expression among tumor and nontumor tissue, revealing limited correlation. This divergence was especially pronounced for species showing biological change, highlighting the complementary perspectives provided by transcriptomics and proteomics. Our work demonstrates the power of metabolic labeling combined with stable isotopic dilution as an integrated strategy for the identification and validation of differentially expressed proteins using rodent models of human disease.

Huttlin, Edward L.; Chen, Xiaodi; Barrett-Wilt, Gregory A.; Hegeman, Adrian D.; Halberg, Richard B.; Harms, Amy C.; Newton, Michael A.; Dove, William F.; Sussman, Michael R.



Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples.  


New stable isotope dilution assays were developed for the simultaneous quantitation of [(13)C(5)]-labelled and unlabelled 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, folic acid along with unlabelled tetrahydrofolic acid and 10-formylfolic acid in clinical samples deriving from human bioavailability studies, i.e. plasma, ileostomy samples, and food. The methods were based on clean-up by strong anion exchange followed by LC-MS/MS detection. Deuterated analogues of the folates were applied as the internal standards in the stable isotope dilution assays. Assay sensitivity was sufficient to detect all relevant folates in the respective samples as their limits of detection were below 0.62 nmol/L in plasma and below 0.73 ?g/100 g in food or ileostomy samples. Quantification of the [(13)C(5)]-label in clinical samples offers the possibility to differentiate between folate from endogenous body pools and the administered dose when executing bioavailability trials. PMID:21052651

Büttner, Barbara E; Öhrvik, Veronica E; Witthöft, Cornelia M; Rychlik, Michael



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



Does a stable isotopically labeled internal standard always correct analyte response?  

Microsoft Academic Search

A stable isotopically labeled (SIL) analogue is believed to be the most appropriate internal standard in a quantitative bioanalytical liquid chromatography\\/tandem mass spectrometry (LC\\/MS\\/MS) assay. It is assumed that a SIL internal standard always compensates for variability in chemical derivatization, sample extraction and LC\\/MS\\/MS analysis due to its nearly identical chemical and physical properties to the unlabeled analyte. Hence, the

Sherry Wang; Matthew Cyronak; Eric Yang



Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover  

Microsoft Academic Search

Tracer methods for VLDL-TG kinetics vary in their ability to account for the effect of tracer recycling, which can influence the calculation of VLDL-TG fractional catabolic rates (FCRs). We evaluated a novel approach, in- volving stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling, for measur- ing VLDL-TG kinetics in normolipidemic human subjects. When administered as a

Bruce W. Patterson; Bettina Mittendorfer; Nizar Elias; Raj Satyanarayana; Samuel Klein


Apolipoprotein B metabolism in humans: studies with stable isotope-labeled amino acid precursors  

Microsoft Academic Search

This article reviews the literature from 1986 to early 2001 relating to apoB100 and apoB48 kinetics in humans using amino acid precursors labeled with stable isotopes. The following subjects are reviewed: (1) methodology; (2) normal individuals and the effects of aging; (3) diet; (4) hereditary dyslipidemias: familial hypercholesterolemia, familial combined hyperlipidemia, cholesteryl ester storage disease, cholesteryl ester transfer protein deficiency,

Julian B. Marsh; Francine K. Welty; Alice H. Lichtenstein; Stefania Lamon-Fava; Ernst J. Schaefer



Evaluation of stereo-array isotope labeling (SAIL) patterns for automated structural analysis of proteins with CYANA.  


Recently we have developed the stereo-array isotope labeling (SAIL) technique to overcome the conventional molecular size limitation in NMR protein structure determination by employing complete stereo- and regiospecific patterns of stable isotopes. SAIL sharpens signals and simplifies spectra without the loss of requisite structural information, thus making large classes of proteins newly accessible to detailed solution structure determination. The automated structure calculation program CYANA can efficiently analyze SAIL-NOESY spectra and calculate structures without manual analysis. Nevertheless, the original SAIL method might not be capable of determining the structures of proteins larger than 50 kDa or membrane proteins, for which the spectra are characterized by many broadened and overlapped peaks. Here we have carried out simulations of new SAIL patterns optimized for minimal relaxation and overlap, to evaluate the combined use of SAIL and CYANA for solving the structures of larger proteins and membrane proteins. The modified approach reduces the number of peaks to nearly half of that observed with uniform labeling, while still yielding well-defined structures and is expected to enable NMR structure determinations of these challenging systems. PMID:16602075

Ikeya, Teppei; Terauchi, Tsutomu; Güntert, Peter; Kainosho, Masatsune



Group-specific primary production based on stable-isotope labeling of phospholipid-derived fatty acids  

Microsoft Academic Search

Stable-isotope labeling of phospholipid-derived fatty acids (PLFAs) is a potentially powerful technique to study group-specific primary production of phytoplankton, as many algal groups possess a specific PLFA composition, and it is relatively simple to measure the isotopic composition of a large number of PLFAs. Experiments with cultured algae showed that differences exist in labeling among PLFAs and between PLFAs and

N. A. Dijkman; H. T. S. Boschker; J. J. Middelburg; J. C. Kromkamp



Stable Isotope Labeling with Amino Acids in Drosophila for Quantifying Proteins and Modifications  

PubMed Central

SUMMARY Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here we present in detail the development of a procedure to incorporate stable isotope labeled amino acids into the fly proteome. In the method of Stable Isotope Labeling with Amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in Drosophila melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.

Xu, Ping; Tan, Huiping; Duong, Duc M.; Yang, Yanling; Kupsco, Jeremy; Moberg, Kenneth H.; Li, He; Jin, Peng; Peng, Junmin



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

PubMed Central

Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for post-measurement normalization of peptide ratios, which is required by the other programs.

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



Synthesis of isotopically labeled P-site substrates for the ribosomal peptidyl transferase reaction  

PubMed Central

Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb,1 have been designed and synthesized in 26–350020steps. These include individual isotopic substitution at the ?-proton, carbonyl carbon, and carbonyl oxygen of the amino acid, the O2' and O3' of the adenosine, and a remote label in the N3 and N4 of both cytidines. These isotopomers were synthesized by coupling cytidylyl-(3'5')-cytidine phosphoramidite isotopomers, as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic enrichment is higher than 99% for 1-13C (Phe), 2-2H (Phe), and 3,4-15N2 (cytidine), 93% for 2'/3'- 18O (adenosine), and 64% for 1-18O (Phe). A new synthesis of highly enriched [1-18O2] phenylalanine has been developed. The synthesis of [3'-18O] adenosine was improved by Lewis acid aided regioselective ring opening of the epoxide and by an economical SN2-SN2 method with high isotopic enrichment (93%). Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes, including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes.

Zhong, Minghong



¹?N metabolic labeling: evidence for a stable isotope effect on plasma protein levels and peptide chromatographic retention times.  


Many quantitative proteomics methods rely on protein and peptide labeling with stable isotopes. We have recently found that the introduction of ¹?N into organisms via in vivo metabolic labeling affects protein expression levels as well as metabolic pathways and behavioral phenotypes. Here, we present further evidence for a stable isotope effect based on the plasma proteome analysis of ¹?N-labeled mice. We compared plasma proteomes of ¹?N-labeled and unlabeled (¹?N) mice by quantitative MS. We found a number of protein level differences, some of which were verified immunochemically. In addition, we observed divergent chromatographic retention time and peak full width at half maximum (FWHM) between ¹?N-labeled and ¹?N tryptic peptides. Our data point toward a systemic effect of the introduction of heavy isotopes in vivo. PMID:23279933

Webhofer, Christian; Zhang, Yaoyang; Brusis, Jan; Reckow, Stefan; Landgraf, Rainer; Maccarrone, Giuseppina; Turck, Christoph W; Filiou, Michaela D



Stable isotope labels as a tool to determine the iron absorption by Peruvian school children from a breakfast meal  

Microsoft Academic Search

Fractional iron absorption from a breakfast meal was determined in Peruvian children employing stable iron isotopes as labels.\\u000a Iron isotopic analysis was performed by the recently developed negative thermal ionization technique for high-precision iron\\u000a isotope ratio measurements using FeF4\\u000a – ions. By increasing the ascorbic acid content of the standard breakfast meal as served within the Peruvian school-breakfast\\u000a program from

T. Walczyk; Lena Davidsson; Nelly Zavaleta; Richard F. Hurrell



Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria  

PubMed Central

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (TEM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni2+ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of 15N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a Kd of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for TEM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of 15N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.

Chang, Shih Chieh; Galea, Charles A.; Leung, Eleanor W W.; Tajhya, Rajeev B.; Beeton, Christine; Pennington, Michael W.; Norton, Raymond S.




Technology Transfer Automated Retrieval System (TEKTRAN)

Four isotopically (13C) labeled phenolic acids (caffeic [M+3], sinapic [M+2], p-coumaric [M+6] and ferulic [M+6] acids) were synthesized via a simple one step malonic acid condensation with a series of aldehydes. The aldehydes and the malonic acid were variously labeled and unlabeled to vary the enr...


Stable isotope labeling and label-free proteomics of Drosophila parkin null mutants  

PubMed Central

Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and formation of intracytoplasmic Lewy bodies (LBs). Loss-of-function mutations in parkin which encodes an E3 ubiqutin protein ligase contribute to a predominant cause of a familial form of PD termed autosomal recessive juvenile Parkinsonism (AR-JP). Drosophila parkin null mutants display muscle degeneration and mitochondrial dysfunction, providing an animal model to study Parkin-associated molecular pathways in PD. To define protein alterations involved in Parkin pathogenesis, we performed quantitative proteomic analyses of Drosophila parkin null mutants and age-matched controls utilizing both global internal standard technology (GIST) and extracted ion chromatogram peak area (XICPA) label-free approaches. A total of 375 proteins were quantified with a minimum of two peptide identifications from the combination of the XICPA and GIST measurements applied to two independent biological replicates. Sixteen proteins exhibited significant alteration. Seven of the dysregulated proteins are involved in energy metabolism, of which six were down-regulated. All five proteins involved in transporter activity exhibited higher levels, of which larval serum protein 1?, larval serum protein 1?, larval serum protein 1?, and fat body protein 1 showed > 10-fold up-regulation and substantially higher level of fat body protein 1 was confirmed by Western blot analysis. These findings suggest that abnormalities in energy metabolism and protein transporter activity pathways may be associated with the pathogenesis of Parkin-associated ARJP.

Xun, Zhiyin; Kaufman, Thomas C.; Clemmer, David E.



Tracing bioavailability of ZnO nanoparticles using stable isotope labeling.  


Zinc oxide nanoparticles (ZnO NPs) are widely used in commercial products and knowledge of their environmental fate is a priority for ecological protection. Here we synthesized model ZnO NPs that were made from and thus labeled with the stable isotope (68)Zn and this enables highly sensitive and selective detection of labeled components against high natural Zn background levels. We combine high precision stable isotope measurements and novel bioimaging techniques to characterize parallel water-borne exposures of the common mudshrimp Corophium volutator to (68)ZnO NPs, bulk (68)ZnO, and soluble (68)ZnCl(2) in the presence of sediment. C. volutator is an important component of coastal ecosystems where river-borne NPs will accumulate and is used on a routine basis for toxicity assessments. Our results demonstrate that ionic Zn from ZnO NPs is bioavailable to C. volutator and that Zn uptake is active. Bioavailability appears to be governed primarily by the dissolved Zn content of the water, whereby Zn uptake occurs via the aqueous phase and/or the ingestion of sediment particles with adsorbed Zn from dissolution of ZnO particles. The high sorption capacity of sediments for Zn thus enhances the potential for trophic transfer of Zn derived from readily soluble ZnO NPs. The uncertainties of our isotopic data are too large, however, to conclusively rule out any additional direct uptake route of ZnO NPs by C. volutator. PMID:23050854

Larner, Fiona; Dogra, Yuktee; Dybowska, Agnieszka; Fabrega, Julia; Stolpe, Björn; Bridgestock, Luke J; Goodhead, Rhys; Weiss, Dominik J; Moger, Julian; Lead, Jamie R; Valsami-Jones, Eugenia; Tyler, Charles R; Galloway, Tamara S; Rehkämper, Mark



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


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-(13)C] 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-(13)C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra. PMID:23990199

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



31P NMR correlation maps of 18O/16O chemical shift isotopic effects for phosphometabolite labeling studies  

PubMed Central

Intramolecular correlations among the 18O-labels of metabolic oligophosphates, mapped by J-decoupled 31P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the 18O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of 18O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological usefulness of the J-decoupled 31P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally 18O labeled in perfused mammalian hearts.

Nemutlu, Emirhan; Zhang, Song; Dzeja, Petras; Terzic, Andre; Macura, Slobodan



Identification of ejaculated proteins in the house mouse (Mus domesticus) via isotopic labeling  

PubMed Central

Background Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins. Results We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract. Conclusion Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract.



Does long contact with the soil improve the efficiency of rock phosphate? Results of isotopic studies  

Microsoft Academic Search

The effect of incubation on the fate of phosphorus in four phosphatic fertilizers (diammonium phosphate and three rock phosphates) applied to four weakly acid to acid soils was studied. Percent utilisation of fertilizer P by the crop was measured by isotopic labelling and the level and quality of available soil P following addition of fertilizer was measured by the isotopic

Jean-Claude Fardeau; Christian Morel; Michel Jahiel



Quantitative proteomics analysis of high-density lipoproteins by stable 18O-isotope labeling.  


For the large-scale study of dynamic proteomes, quantitative proteomic approaches based on stable isotope labeling and mass spectrometry (MS) have been developed as a high-throughput, reproducible, and robust alternative to conventional gel-based techniques. In this chapter, we describe in detail a quantitative proteomic strategy based on HDL isolation by affinity chromatography, in-gel trypsin digestion of protein extracts, peptide (18)O labeling, separation by off-gel isoelectric focusing, and peptide analysis on a linear ion trap mass spectrometer, followed by the application of a robust multilayered statistical model. This protocol is of universal applicability and has been successfully applied to the global characterization of the HDL proteome with some specific considerations for this particle, paving the way to the in-depth study of the protein cargo of HDL and its implication in cardiovascular diseases. PMID:23585090

Burillo, Elena; Vazquez, Jesus; Jorge, Inmaculada



Examination of the exchange interaction through micelle size. 2. Isotope separation efficiency as an experimental probe  

SciTech Connect

The geminate reaction probabilities (for recombination and disproportionation) of benzoyl/sec-phenethyl radical pairs, generated by the photolysis of [alpha]-methyldeoxybenzoin, for both unlabeled ([sup 13]C in natural abundance at the carbonyl position) and labeled ketones ([sup 13]C in the carbonyl position) were measured in different sized alkyl sulfate micelles (sodium octyl sulfate (C[sub g]) through sodium dodecyl sulfate (C[sub 12])) in zero and high magnetic fields (B = 2400 G). Although the probability of geminate recombination (P[sub t]) diminishes for the unlabeled pair, from 0.549 to 0.436 and for the labeled pair from 0.585 to 0.504 at zero magnetic field with decreasing micelle size (C[sub 12] to C[sub 8]), the efficiency of isotope separation ([alpha]) is found to increase at zero magnetic field from 1.144 to 1.236 with decreasing micelle size. Theoretical considerations of these experimental results show that the rate of geminate reaction of the unlabeled radical pairs in small micelles is sensitive to the electron spin exchange interaction; intersystem crossing is influenced by fast forced reencounters. These effects are not as important for the labeled radical pairs (which possess a strong [sup 13]C hyperfine interaction). 40 refs., 6 figs., 3 tabs.

Tarasov, V.F.; Buchachenko, A.L. (Inst. of Chemical Physics, Moscow (Russian Federation)); Ghatlia, N.D.; Turro, N.J. (Columbia Univ., New York, NY (United States)); Avdievich, N.I. (International Tomography Center, Novosibirsk (Russian Federation)); Shkrob, I.A. (Argonne National Lab., IL (United States))



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



Carbon allocation belowground in Pinus pinaster using stable carbon isotope pulse labeling technique  

NASA Astrophysics Data System (ADS)

Carbon allocation belowground competes with aboveground growth and biomass production. In the other hand, it contributes to resource acquisition such as nutrient, water and carbon sequestration in soil. Thus, a better characterization of carbon flow from plant to soil and its residence time within each compartment is an important issue for understanding and modeling forest ecosystem carbon budget. 13C pulse labeling of whole crown was conducted at 4 seasons to study the fate of assimilated carbon by photosynthesis into the root on 12 year old Pinus pinaster planted in the INRA domain of Pierroton. Maritime pine is the most widely planted species in South-West Europe. Stem, root and soil CO2 effluxes and their isotope composition were measured continuously by tunable diode laser absorption spectroscopy with a trace gas analyzer (TGA 100A; Campbell Scientific) coupled to flow-through chambers. 13CO2 recovery and peak were observed in respiration of each compartment after labeling. It appeared sequentially from top of stem to bottom, and to coarse root. The maximum velocity of carbon transfer was calculated as the difference in time lag of recovery between two positions on the trunk or on the root. It ranged between 0.08-0.2 m h-1 in stem and between 0.04-0.12 m h-1 in coarse root. This velocity was higher in warmer season, and the difference between time lag of recovery and peak increased after first frost. Photosynthates arrived underground 1.5 to 5 days after labeling, at similar time in soil CO2 effluxes and coarse root respiration. 0.08-1.4 g of carbon was respired per tree during first 20 days following labeling. It presented 0.6 -10% of 13C used for labeling and it is strongly related to seasons. The isotope signal was detected in fine root organs and microbial biomass by periodical core sampling. The peak was observed 6 days after labeling in early summer while it was delayed more than 10 days in autumn and winter with less amount of carbon allocated belowground.

Dannoura, M.; Bosc, A.; Chipeaux, C.; Sartore, M.; Lambrot, C.; Trichet, P.; Bakker, M.; Loustau, D.; Epron, D.



Isotope labeling studies on the electron impact mass spectral fragmentation patterns of chloropropanol acetates.  


Chloropropanol (CP) esters are part of an emerging group of process-induced toxicants that are considered as potential health hazards particularly in palm oil. Mass spectrometry-based methodologies for identification of CP esters in food are critical in overcoming the challenges associated with direct detection methods. In the present study, a convenient strategy was employed to generate all possible CP acetates through reacting acetic anhydride with either glycerol in the presence of a chloride source or the corresponding CPs, such as 3-chloro-, 1,3-dichloro-, 2-chloro-, and 1,2-dichloropropanols, allowing for the identification of the individual CP acetates and assignment of their mass spectral fragmentations. Mass spectral fragmentations were confirmed through the use of the isotopic signature of chlorine in addition to the isotope labeling experiments performed using isotopically labeled precursors, such as [(13)C-U3] glycerol, [(13)C-U4] acetic anhydride, [(13)C-2,2'] acetic anhydride, and [d5] 3-monochloropropane-1,2-diol (3-MCPD) as reactants. Such studies have indicated that all CP esters can undergo two general fragmentations under electron impact (EI) conditions, one generating the acylium ion at m/z 45 and the other generating a chlorinated cyclic acyloxonium ion at m/z 135.6. Considering the fact that such ions can also be generated from any fatty acid containing CP esters after undergoing McLafferty rearrangement, the ion at m/z 135.6 can therefore be considered as a universal marker for the presence of CP esters undergoing EI fragmentation. Furthermore, these studies have also indicated the formation of ions characteristic of CP diesters, monoesters, and dichloro esters. PMID:23964824

Rahn, Anja K K; Yaylayan, Varoujan A



Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.  

PubMed Central

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

Simplaceanu, V; Lukin, J A; Fang, T Y; Zou, M; Ho, N T; Ho, C



Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol  

PubMed Central

Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol’s well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account.

Lu, Kun; Gul, Husamettin; Upton, Patricia B.; Moeller, Benjamin C.; Swenberg, James A.



Genetic differences in water-use efficiency, stomatal conductance and carbon isotope fractionation in potato  

Microsoft Academic Search

Summary  Potato (Solanum tuberosum L.) cultivars were grown in pots and containers under a rain shelter to examine differences in stomatal conductance, water-use\\u000a efficiency, and carbon isotope fractionation. Conductance was measured on abaxial leaf surfaces with a steady state diffusion\\u000a porometer. Carbon isotopic analyses were made with an isotope ratio mass spectrometer. Water-use efficiency (WUE) was obtained\\u000a by dividing total dry

J. Vos; J. Groenwold



Protein global fold determination using site-directed spin and isotope labeling.  

PubMed Central

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 A were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 A from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.

Gaponenko, V.; Howarth, J. W.; Columbus, L.; Gasmi-Seabrook, G.; Yuan, J.; Hubbell, W. L.; Rosevear, P. R.



Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies  

NASA Astrophysics Data System (ADS)

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.



Respiratory Carbon Metabolism following Illumination in Intact French Bean Leaves Using 13C/12C Isotope Labeling1  

PubMed Central

The origin of the carbon atoms in the CO2 respired by French bean (Phaseolus vulgaris) leaves in the dark has been studied using 13C/12C isotopes as tracers. The stable isotope labeling was achieved through a technical device that uses an open gas-exchange system coupled online to an elemental analyzer and linked to an isotope ratio mass spectrometer. The isotopic analysis of the CO2 respired in the dark after a light period revealed that the CO2 was labeled, but the labeling level decreased progressively as the dark period increased. The pattern of disappearance depended on the amount of carbon fixed during the labeling and indicated that there were several pools of respiratory metabolites with distinct turnover rates. We demonstrate that the carbon recently assimilated during photosynthesis accounts for less than 50% of the carbon in the CO2 lost by dark respiration and that the proportion is not influenced by leaf starvation in darkness before the labeling. Therefore, most of the carbon released by dark respiration after illumination does not come from new photosynthates.

Nogues, Salvador; Tcherkez, Guillaume; Cornic, Gabriel; Ghashghaie, Jaleh



Effect of acetaminophen on the leukocyte-labeling efficiency of indium oxine In 111  

SciTech Connect

The effect of acetaminophen on the labeling efficiency of leukocytes with indium oxine In 111 was studied. A blood sample was obtained from eight healthy men before and after they received acetaminophen 650 mg every four hours for 24 hours. After dividing the plasma from each sample into three portions, leukocytes were separated and labeled with indium oxine In 111. In an in vitro study, 200 ml of blood was obtained from one of the men, and the plasma was separated into four portions. Acetaminophen in 95% ethanol was added to three of the plasma fractions to produce acetaminophen concentrations of 4, 20, and 100 micrograms/ml; ethanol was added to the fourth fraction as a control. Each plasma fraction was then subdivided into three aliquots, and leukocytes were labeled as in the in vivo study. Mean leukocyte labeling efficiencies in both studies were calculated from the ratios of leukocyte radioactivity to initial radioactivity in the samples, expressed as percentages. Leukocyte labeling efficiencies before acetaminophen administration ranged from 79 to 85%; after administration, labeling efficiencies ranged from 70 to 87%. No significant differences in mean labeling efficiency before and after acetaminophen administration were noted in any of the subjects. Leukocyte labeling efficiencies in all in vitro plasma fractions were reduced, ranging from 54 to 63%, but no significant differences in labeling efficiency between any of the plasma fractions were found. Using the labeling procedures in this study, exposure of leukocytes from healthy men to acetaminophen in vivo or in vitro does not affect labeling efficiency with indium oxine In 111.

Augustine, S.C.; Schmelter, R.F.; Nelson, K.L.; Petersen, R.J.; Qualfe, M.A.



Metabolic flux analysis in Synechocystis using isotope distribution from 13C-labeled glucose.  


Using the carbon isotope labeling technique, the response of cyanobacterial central carbon metabolism to the change in environmental conditions was investigated. Synechocystis was grown in the heterotrophic and mixotrophic cultures fed with 13C-labeled glucose. The labeling patterns of the amino acids in biomass hydrolysates for both cultures were detected by the two-dimensional 1H-13C correlation nuclear magnetic resonance (2D 1H-13C COSY NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) technique. The in vivo intracellular flux distributions were then quantitated from the labeling measurements and metabolite balances using a parameters fitting approach. From the estimated flux distributions, it was found that the pentose phosphate pathway was the major pathway of glucose catabolism in the heterotrophic culture, while in the mixotrophic culture, the flux of CO2 fixation through the Calvin cycle was about two-fold of the glucose input flux. The relative flux through the phosphoenolpyruvate carboxylase was very high in both cultures, and this reaction represented about 25% of the assimilated CO2 in the mixotrophic culture. More importantly, we found a substantial outflow from the tricarboxylic acid cycle to glycolysis pathway carried by the malic enzyme, demonstrating the operation of a C4 pathway in cyanobacterial cells through the PEP carboxylase and malic enzyme. The estimated flux distributions also revealed that the NADPH synthesis was in excess relative to its requirement, and the excess NADPH might be reoxidized in cyanobacterial respiration to provide the energy for cellular requirement. Moreover, the analyzed result also suggested that the activity of the respiratory electron transport chain in cyanobacterial cells was not inhibited by light. PMID:12616690

Yang, Chen; Hua, Qiang; Shimizu, Kazuyuki



Closing the gap between T-cell life span estimates from stable isotope-labeling studies in mice and humans.  


Quantitative knowledge of the turnover of different leukocyte populations is a key to our understanding of immune function in health and disease. Much progress has been made thanks to the introduction of stable isotope labeling, the state-of-the-art technique for in vivo quantification of cellular life spans. Yet, even leukocyte life span estimates on the basis of stable isotope labeling can vary up to 10-fold among laboratories. We investigated whether these differences could be the result of variances in the length of the labeling period among studies. To this end, we performed deuterated water-labeling experiments in mice, in which only the length of label administration was varied. The resulting life span estimates were indeed dependent on the length of the labeling period when the data were analyzed using a commonly used single-exponential model. We show that multiexponential models provide the necessary tool to obtain life span estimates that are independent of the length of the labeling period. Use of a multiexponential model enabled us to reduce the gap between human T-cell life span estimates from 2 previously published labeling studies. This provides an important step toward unambiguous understanding of leukocyte turnover in health and disease. PMID:23945154

Westera, Liset; Drylewicz, Julia; den Braber, Ineke; Mugwagwa, Tendai; van der Maas, Iris; Kwast, Lydia; Volman, Thomas; van de Weg-Schrijver, Elise H R; Bartha, István; Spierenburg, Gerrit; Gaiser, Koos; Ackermans, Mariëtte T; Asquith, Becca; de Boer, Rob J; Tesselaar, Kiki; Borghans, José A M



Stable Isotope Labeling Strategy for Protein-Ligand Binding Analysis in Multi-Component Protein Mixtures  

NASA Astrophysics Data System (ADS)

Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H{2/16}O2 and H{2/18}O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

DeArmond, Patrick D.; West, Graham M.; Huang, Hai-Tsang; Fitzgerald, Michael C.



Stable isotope labeling strategy for protein-ligand binding analysis in multi-component protein mixtures.  


Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H(2) (16)O(2) and H(2) (18)O(2) labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the (18)O/(16)O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A). PMID:21472561

DeArmond, Patrick D; West, Graham M; Huang, Hai-Tsang; Fitzgerald, Michael C



Use of Carbon-13 IN Biosynthetic Studies. Incorporation of Isotopically Labelled Acetate and Formate into the Fungal Tropolone, Sepedonin.  

National Technical Information Service (NTIS)

When carbon-13 is used as the isotopic tracer in biosynthetic studies, the labelled atoms in the metabolite can often be determined by n.m.r. spectrometry, without need for chemical degradation. By this technique the biosynthesis of sepedonin in cultures ...

A. G. McInnes D. G. Smith L. C. Vining J. L. C. Wright



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.



Method to Test Isotopic Separation Efficiency of Palladium Packed Columns.  

National Technical Information Service (NTIS)

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 ...

J. C. Staack J. E. Klein L. K. Heung W. D. Jacobs



Effect of acetaminophen on the leukocyte-labeling efficiency of indium oxine In 111  

Microsoft Academic Search

The effect of acetaminophen on the labeling efficiency of leukocytes with indium oxine In 111 was studied. A blood sample was obtained from eight healthy men before and after they received acetaminophen 650 mg every four hours for 24 hours. After dividing the plasma from each sample into three portions, leukocytes were separated and labeled with indium oxine In 111.

S. C. Augustine; R. F. Schmelter; K. L. Nelson; R. J. Petersen; M. A. Qualfe



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)



Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope assisted labeling (SIAL)  

PubMed Central

OBJECTIVE Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, diabetes mellitus type II, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. METHODS We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy, and cavity ringdown spectroscopy to analyze serial plasma samples and real-time breath measurements following selective 13C-isotope assisted labeling (SIAL). These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. RESULTS Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. CONCLUSION This novel diagnostics approach is fast, non-invasive and sensitive for determining specific pathway utilization, and provides a direct translational application in the healthcare field.

Haviland, Julia A.; Tonelli, Marco; Haughey, Dermot T.; Porter, Warren P.; Assadi-Porter, Fariba M.



Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients  

PubMed Central

Background Dengue fever is the most important arthropod born viral disease of public health significance. Although most patients suffer only from flu-like symptoms, a small group of patient experiences more severe forms of the disease. To contribute to a better understanding of its pathogenesis this study aims to identify proteins differentially expressed in a pool of five viremic plasma from severe dengue patients relative to a pool of five non-severe dengue patients. Results The use of Isotope Coded Protein Labeling (ICPLTM) to analyze plasma depleted of twenty high-abundance proteins allowed for the identification of 51 differentially expressed proteins, which were characterized by mass spectrometry. Using quantitative ELISA, three of these proteins (Leucine-rich glycoprotein 1, Vitamin D binding-protein and Ferritin) were confirmed as having an increased expression in a panel of severe dengue plasma. The proteins identified as overexpressed by ICPLTM in severe dengue plasma involve in clear up action after cell injury, tissue coherence and immune defense. Conclusion This ICPLTM study evaluating differences between acute severe dengue plasmas and acute non-severe dengue plasmas suggests that the three proteins identified are overexpressed early in the course of the disease. Their possible use as biomarkers for the prognostic of disease severity is discussed.



Synthesis of stable isotope labeled analogs of the anti-hepatitis C virus nucleotide prodrugs PSI-7977 and PSI-352938.  


In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent (13)CD(3)MgI to the corresponding 2 '-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 '-C-(trideuterated-(13)C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones. PMID:22060553

Chun, Byoung-Kwon; Du, Jinfa; Zhang, Hai-Ren; Chang, Wonsuk; Ross, Bruce S; Jiang, Ying; Bao, Donghui; Espiritu, Christine L; Keilman, Meg; Steuer, Holly M Micolochick; Furman, Phillip A; Sofia, Michael J



Comparing the Efficiencies of Hydrazide Labels in the Study of Protein Carbonylation in Human Serum Albumin  

PubMed Central

In this work, we establish a methodology for comparing the efficiencies of different hydrazide labels for detecting protein carbonyls. We have chosen acrolein- modified human serum albumin as a model. This system provides a convenient means of reproducibly generating carbonylated protein. Five hydrazide-based labels were tested. Three carry a biotin affinity tag and the others are simple fatty acid hydrazides. For the biotin-based labels, the yield of the labeling reaction varies considerably and the most commonly used label, biotin hydrazide, gives the lowest yield. The total MS/MS spectrum counts of modified peptides are similar for all of the biotin-based tags indicating that factors beyond the labeling efficiency are important in determining the effectiveness of the label. In addition, there is a large variation in the number of spectra obtained for specific, modified peptides depending on the nature of the labeling group. This variation implies that the relative dectability of a particular modification site is highly dependent on the tagging reagent, and more importantly, titration schemes aimed at identifying the most reactive site based on its threshold concentration will be biased by the choice of tagging reagent. The fatty acid hydrazides are somewhat more effective than the biotin-based hydrazides in generating identifiable MS/MS spectra, but offer no opportunity for enrichment. For the biotin-based tags, avidin affinity chromatography was used with the tryptic digests and each tag led to similar enrichment levels.

Ugur, Zafer; Coffey, Chelsea M.; Gronert, Scott



Phosphorus use efficiency by cotton measured through 32P isotope technique  

NASA Astrophysics Data System (ADS)

Deficiency of phosphorus (P) is the major limitation to agricultural production in the Brazilian Savannah (Cerrado), which is naturally poor in this nutrient. Most of the P applied by fertilizer in Cerrado soils are converted into low solubility forms and can not be easily absorbed by plants. This occurs for characteristics of adsorption, conditioned by the predominance of low pH and aluminum and iron oxides in the clay fraction. The development of genotypes and cultivars with greater capacity to grow up in soils with low P availability ('phosphorus efficiency') is interesting to improve the agriculture in these areas in a sustainable way. Cotton (Gossypium spp.) is the main product for the fibers used nationally and globally in the textile chain. This study aim was to evaluate the efficiency of absorption and utilization of P by cotton cultivars/genotypes grown in Cerrado soil by the isotopic dilution technique. The soil classified as Ultisols, was labeled with the radioisotope 32P.The experiment was conducted in a greenhouse in a completely randomized design factorial 2 x 17. Factors were considered two levels of P (insufficient = 20 mg kg-1 and sufficient = 120 mg kg-1) and 17 genetic materials of cotton recommended for Cerrado region. Phosphorus levels influenced significantly the shoots dry matter production, the P content and accumulation, the 32P specific activity, the L value and L value less seed cotton P by cultivars and genotypes. The hierarchical clustering analysis used to verify the similarities between the cultivars and genotypes of cotton, classified them into internally homogeneous groups and heterogeneous between different groups. Cultivars FMT 523, FM 910 and CNPA GO 2043 were the most responsive to phosphate fertilizer in sufficient level of P, while the genotype Barbadense 01 and cultivars FM 966LL, IPR Jataí, BRS Aroeira and BRS Buriti were most efficient absorbing P in soils with insufficient level.

Marcante, N. C.; Muraoka, T.; Camacho, M. A.; César, F. R. C. F.; Bruno, I. P.



Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways  

Microsoft Academic Search

BackgroundMetabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling

Eisuke Chikayama; Michitaka Suto; Takashi Nishihara; Kazuo Shinozaki; Takashi Hirayama; Jun Kikuchi; Lucia Banci



Application of a mechanistic model of bovine milk protein synthesis to examine the use of isotope labeling methods.  


Two types of models of bovine milk protein synthesis were used to simulate collection and analysis of data from infusion experiments involving isotope-labeled amino acids (AA). Analytical solutions to a system of ordinary differential equations that describe isotope enrichment curves of each AA pool within the mammary gland were derived and are presented. Numerical solutions from a dynamic mechanistic model suggest that normal experimental procedures can affect the shape of enrichment curves and, therefore, results derived from them. Simulation results suggest that standard methods utilizing in vivo isotope kinetics may be of limited value to characterize the metabolism of the bovine mammary gland, especially AA metabolism and milk protein synthesis and secretion. The results clearly demonstrate the flexibility of such models for the testing of many hypotheses and possible experimental protocols. PMID:9785235

Maas, J A; France, J; McBride, B W



A SILAC compatible strain of Pichia pastoris for expression of isotopically labeled protein standards and quantitative proteomics  

PubMed Central

The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications, and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.

Austin, Ryan J.; Kuestner, Rolf E.; Chang, Debbie K.; Madden, Knut R.; Martin, Daniel B.



The effect of pregnancy in humans on the pharmacokinetics of stable isotope labelled phenytoin.  

PubMed Central

1. To investigate the mechanism of the fall in steady-state plasma phenytoin concentration relative to drug dose that occurs during pregnancy, single dose pharmacokinetic studies with stable isotope labelled phenytoin were carried out at different stages of pregnancy, and 2 to 4 months post-natally, in five epileptic women receiving regular oral therapy with the drug. 2. Steady-state apparent plasma clearances of phenytoin (dose/steady-state concentration) correlated closely with simultaneous plasma clearances of the intravenous stable-isotope drug (measured as dose/AUC) suggesting that the patients were complaint with therapy when their phenytoin dosage requirement increased during the pregnancy, and that the oral drug was fully bioavailable. 3. In retrospect, two of the five subjects were probably studied too early post-natally for phenytoin elimination kinetics to have returned to non-pregnant values. Despite this, (i) the mean +/- s.d. t 1/2 for phenytoin was statistically significantly shorter in pregnancy than post-natally (31 +/- 14 vs 39 +/- 28 h), (ii) the mean +/- s.d. whole plasma clearance was also statistically significant greater (0.025 +/- 0.012 vs 0.021 +/- 0.013 kg-1 h-1) and (iii) the mean +/- s.d. Vmax for phenytoin elimination was statistically significantly greater in pregnancy (1170 +/- 600 mg day-1) than post-natally (780 +/- 470 mg day-1). Although the mean +/- s.d. apparent Km was higher in pregnancy (18.2 +/- 8.4 mg l-1, expressed in terms of whole plasma drug concentrations, compared with 10.2 +/- 7.4 mg l-1 post-natally), the difference was not statistically significant. However, if the apparent Km value was expressed in terms of plasma water phenytoin concentrations the difference (pregnant 2.50 +/- 0.85 mg l-1: post-natally 1.16 +/- 0.65 mg l-1) was statistically significant. 4. Human pregnancy appears to result in an increased capacity to eliminate phenytoin.

Dickinson, R G; Hooper, W D; Wood, B; Lander, C M; Eadie, M J



Application of isotopically labeled methylmercury for isotope dilution analysis of biological samples using gas chromatography/ICPMS.  


An isotope dilution (ID) procedure for the determination of methylmercury (MMHg) in biological samples using an inductively coupled plasma mass spectrometer as detector after the capillary gas chromatographic separation (CGC/ICPMS) has been developed. For the first time, open-focused-microwave pretreatment has been used in conjunction with ID. Optimum conditions for the measurement of isotope ratios on the fast transient chromatographic peaks have been established. Mass bias was found to be about 1.5%/mass unit and was corrected by using the simultaneously measured thallium signals at 203Tl and 205Tl. After mass-bias correction, deviation of the theoretical mercury ratio values was found to be as low as 0.2%. Isotope ratio precisions based on the peak areas measurements were 0.3% RSD for 20 pg injected (as Hg absolute). The absolute detection limits were in the range of 20-30 fg for 202Hg and 201Hg. Methylmercury enriched in 201Hg has been synthesized by direct reaction with methylcobalamine. The concentration of the MMHg spike has been measured by reverse isotope dilution with a natural MMHg standard. The capabilities of CGC/ICPMS to measure isotope ratios were used to optimize sample derivatization by aqueous ethylation with NaBEt4 with respect to MMHg degradation pathways and quantitative recovery. The accuracy of the method developed has been validated with biological certified reference materials (CRM-463, DORM-1). PMID:12069230

Rodriguez Martín-Doimeadios, R C; Krupp, E; Amouroux, D; Donard, O F X



Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.  


Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency. PMID:22648530

Wang, Chen; Ouyang, Jun; Ye, De-Kai; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua



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



Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability.  


By complexing ferumoxides or superparamagnetic iron oxide (SPIO) to transfection agents (TAs), it is possible to magnetically label mammalian cells. There has been no systematic study comparing TAs complexed to SPIO as far as cell labeling efficiency and viability. This study investigates the toxicity and labeling efficiency at various doses of FEs complexed to different TAs in mammalian cells. Different classes of TAs were used, such as polycationic amines, dendrimers, and lipid-based agents. Cellular toxicity was measured using doses of TAs from 1 to 50 microg/mL in incubation media. Iron incorporation efficiency was measured by combining various amounts of FEs and different doses of TAs. Lipofectamine2000 showed toxicity at lowest dose (1 microg/mL), whereas FuGENE6 and low molecular weight poly-L-lysine (PLL) showed the least toxicity. SPIO labeling efficiency was similar with high-molecular-weight PLL (388.1 kDa) and superfect, whereas FuGENE6 and low-molecular-weight PLL were inefficient in labeling cells. Concentrations of 25 to 50 microg/mL of FEs complexed to TAs in media resulted in sufficient endocytosis of the SPIO into endosomes to detect cells on cellular magnetic resonance imaging. PMID:15142409

Arbab, Ali Syed; Yocum, Gene Thomus; Wilson, Lindsey Bashaw; Parwana, Ashari; Jordan, Elaine Kay; Kalish, Heather; Frank, Joseph Alan



Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants  

PubMed Central

We describe the use of carbon stable isotope (13C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically 13C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha?1) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the ?13C (i.e. the ratio 13C:12C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C27–C36) and passage kinetics were determined for the most abundant C29, C31 and C33 n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K1) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p?0.002) increase with carbon chain length. K1 estimates were comparable to those of the 13C labeled digestible dry matter fraction (3.38%/h; r?=?0.61 to 0.71; p?0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of 13C versus 12C. Our results suggest that 13C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the ?13C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics.

Warner, Daniel; Ferreira, Luis M. M.; Breuer, Michel J. H.; Dijkstra, Jan; Pellikaan, Wilbert F.



Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants.  


We describe the use of carbon stable isotope ((13)C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically (13)C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha(-1)) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the ?(13)C (i.e. the ratio (13)C:(12)C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C27-C36) and passage kinetics were determined for the most abundant C29, C31 and C33 n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K 1) among individual n-alkanes (3.71-3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p?0.002) increase with carbon chain length. K 1 estimates were comparable to those of the (13)C labeled digestible dry matter fraction (3.38%/h; r?=?0.61 to 0.71; p?0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of (13)C versus (12)C. Our results suggest that (13)C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the ?(13)C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics. PMID:24124493

Warner, Daniel; Ferreira, Luis M M; Breuer, Michel J H; Dijkstra, Jan; Pellikaan, Wilbert F



Investigation of non-segregation graphene growth on Ni via isotope-labeled alcohol catalytic chemical vapor deposition  

NASA Astrophysics Data System (ADS)

Here we present CVD growth of graphene on Ni and investigate the growth mechanism using isotopically labeled 13C-ethanol as the precursor. Results show that during low-pressure alcohol catalytic CVD (LP-ACCVD), a growth time of less than 30 s yields graphene films with high surface coverage (>80%). Moreover, when isotopically labeled ethanol precursors were sequentially introduced, Raman mapping revealed that both 12C and 13C graphene flakes exist. This shows that even at high temperature (~900 °C) the graphene flakes form independently, suggesting a different growth mechanism for ethanol-derived graphene on Ni from the segregation process for methane-derived graphene. We interpret this growth mechanism using a direct surface-adsorptive growth model in which small carbon fragments catalyzed from ethanol decomposition products first nucleate at metal step edges or grain boundaries to initiate graphene growth, and then expand over the entire metal surface.Here we present CVD growth of graphene on Ni and investigate the growth mechanism using isotopically labeled 13C-ethanol as the precursor. Results show that during low-pressure alcohol catalytic CVD (LP-ACCVD), a growth time of less than 30 s yields graphene films with high surface coverage (>80%). Moreover, when isotopically labeled ethanol precursors were sequentially introduced, Raman mapping revealed that both 12C and 13C graphene flakes exist. This shows that even at high temperature (~900 °C) the graphene flakes form independently, suggesting a different growth mechanism for ethanol-derived graphene on Ni from the segregation process for methane-derived graphene. We interpret this growth mechanism using a direct surface-adsorptive growth model in which small carbon fragments catalyzed from ethanol decomposition products first nucleate at metal step edges or grain boundaries to initiate graphene growth, and then expand over the entire metal surface. Electronic supplementary information (ESI) available: Additional Raman imaging maps from as-grown (isotopic) graphene samples, as well as AFM images of Ni surfaces before and after H2 annealing. See DOI: 10.1039/c3nr01080e

Zhao, Pei; Hou, Bo; Chen, Xiao; Kim, Sungjin; Chiashi, Shohei; Einarsson, Erik; Maruyama, Shigeo



Platelet labeling in plasma made simple and efficient: Preparation and evaluation  

SciTech Connect

Over the past few years the use of In-111 platelets (In-111-P) has continued to grow rapidly. The lack of efficient labeling in plasma have made the preparation procedures complex resulting into almost individualized methods which have used a variety of salt balance media, anticoagulants, and centrifugal forces, and thereby made the comparison of results from different laboratories impossible. The authors have developed a kit method for preparing In-111-P in plasma that restricts these parameters, uses 2 of dry Mercaptopyridine-N-oxide (Merc) or its Na-salt and a weak chelate of In-111 either in solution at pH 6-6.5 or in a dry form. Eighty to 95% labeling efficiency is achieved in 20 minutes incubation at an ambient temperature. Seventy percent radioactivity is incorporated within two minutes, 90% of which is bound to cytoplasmic components. The aggregability of In-111-P remains practically unchanged. In a caning model, In-111-P thus prepared, had 65% recovery and 7.5 days survival. Two - 50 of oxine or tropolone employed under identical conditions produced only 4%-28% and 7%-10% labeling efficiency. Similarly, with 2 Merc, labeling efficiency using Ga-67, Tc-99m, Tl-201, and I-131 was only 7.5%, 6.8%, 13.8% and 5.8% respectively. This new approach provides investigators a simple, efficient and uniform method for preparing viable In-111-P in plasma.

Thakur, M.L.; McKenney, S.; Park, C.H.; Philp, M.S.; Bruggman, T.; Werre, G.



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.



Polyamidoamine dendrimer-conjugated quantum dots for efficient labeling of primary cultured mesenchymal stem cells.  


Monitoring of cells in vivo after transplantation could supply important information for determining the efficacy of stem cell therapy. The use of quantum dots (QDs) has several advantages for in vivo imaging, such as remarkable resistance to photo bleaching, high fluorescence efficiency, and size-tunable emission. After they are taken up by cells via endocytosis, QDs lose their fluorescence intensity in endosomes/lysosomes at low pH because the intensity cannot survive under acidic conditions. Moreover, the amount of QD uptake by mesenchymal stem cells (MSCs) is extremely small. Therefore, for effective labeling of MSCs and long observation of MSCs labeled by QDs in vivo, it is essential both to increase cellular uptake of QDs and to promote endosomal escape into the cytosol. The polyamidoamine (PAMAM) dendrimer had plenty of cationic charge, which promoted cellular uptake though electrostatic interactions, and a "buffering capacity," which enhanced endosomal escape into the cytosol. In this study, QDs were modified with PAMAM dendrimer for the efficient labeling of MSCs by QDs. The uptake efficiency and cytosolic distribution of QDs in primary cultured MSCs were increased by the modification of the PAMAM dendrimer. The fluorescence intensity in MSCs labeled by PAMAM dendrimer-conjugated QDs lasted for a longer time in harvested culture plates or in cell-transplanted mice than that in MSCs labeled by non-conjugated QDs. PMID:21700331

Higuchi, Yuriko; Wu, Can; Chang, Kai-Ling; Irie, Kei; Kawakami, Shigeru; Yamashita, Fumiyoshi; Hashida, Mitsuru



Development And Evaluation Of Stable Isotope And Fluorescent Labeling And Detection Methodologies For Tracking Injected Bacteria During In Situ Bioremediation  

SciTech Connect

This report summarizes the results of a research project conducted to develop new methods to label bacterial cells so that they could be tracked and enumerated as they move in the subsurface after they are introduced into the groundwater (i.e., during bioaugmentation). Labeling methods based on stable isotopes of carbon (13C) and vital fluorescent stains were developed. Both approaches proved successful with regards to the ability to effectively label bacterial cells. Several methods for enumeration of fluorescently-labeled cells were developed and validated, including near-real time microplate spectrofluorometry that could be performed in the field. However, the development of a novel enumeration method for the 13C-enriched cells, chemical reaction interface/mass spectrometry (CRIMS), was not successful due to difficulties with the proposed instrumentation. Both labeling methodologies were successfully evaluated and validated during laboratory- and field-scale bacterial transport experiments. The methods developed during this research should be useful for future bacterial transport work as well as other microbial ecology research in a variety of environments. A full bibliography of research articles and meeting presentations related to this project is included (including web links to abstracts and full text reprints).

Mark E. Fuller; Tullis C. Onstott



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



High efficiency Hall effect micro-biosensor platform for detection of magnetically labeled biomolecules  

Microsoft Academic Search

Detection of magnetically labeled biomolecules using micro-Hall biosensors is a promising method for monitoring biomolecular recognition processes. The measurement efficiency of standard systems is limited by the time taken for magnetic beads to reach the sensing area of the Hall devices. Here, micro-current lines were integrated with Hall effect structures to manipulate the position of magnetic beads via field gradients

Adarsh Sandhu; Yoshimichi Kumagai; Adam Lapicki; Satoshi Sakamoto; Masanori Abe; Hiroshi Handa



Status of China's Energy Efficiency Standards and Labels for Appliances and International Collaboration  

SciTech Connect

China first adopted minimum energy performance standards (MEPS) in 1989. Today, there are standards for a wide range of domestic, commercial and selected industrial equipment. In 1999, China launched a voluntary endorsement label, which has grown to cover over 40 products including water-saving products (See Figure 1). Further, in 2005, China started a mandatory energy information label (also referred to as the 'Energy Label'). Today, the Energy Label is applied to four products including: air conditioners; household refrigerators; clothes washers; and unitary air conditioners (See Figure 2). MEPS and the voluntary endorsement labeling specifications have been updated and revised in order to reflect technology improvements to those products in the market. These programs have had an important impact in reducing energy consumption of appliances in China. Indeed, China has built up a strong infrastructure to develop and implement product standards. Historically, however, the government's primary focus has been on the technical requirements for efficiency performance. Less attention has been paid to monitoring and enforcement with a minimal commitment of resources and little expansion of administrative capacity in this area. Thus, market compliance with both mandatory standards and labeling programs has been questionable and actual energy savings may have been undermined as a result. The establishment of a regularized monitoring system for tracking compliance with the mandatory standard and energy information label in China is a major area for program improvement. Over the years, the Collaborative Labeling and Appliance Standards Program (CLASP) has partnered with several Chinese institutions to promote energy-efficient products in China. CLASP, together with its implementing partner Lawrence Berkeley National Laboratory (LBNL), has assisted China in developing and updating the above-mentioned standards and labeling programs. Because of the increasing need for the development of a monitoring system to track compliance with standards and labeling, CLASP, with support from Japan's Ministry of Economy, Trade and Industry (METI), has expanded its ongoing collaboration with the China National Institute of Standards (CNIS) to include enforcement and monitoring. CNIS has already begun working on the issue of compliance. CNIS has conducted modest sample testing in 2006 for refrigerators, freezers and room air-conditioners, and repeated the same task in 2007 with a similar sample size for three products (refrigerators, freezers, air-conditioners and clothes washers). And, CNIS, with technical support from LBNL, has analyzed the data collected through testing. At the same time, parallel effort has also been paid to look at the potential impact of the label to 2020. In conjunction with CNIS, CLASP technical experts reviewed the standards development timeline of the four products currently subject to the mandatory energy information label. CLASP, with the support of METI/IEEJ, collaborated with CNIS to develop the efficiency grades, providing: technical input to the process; comment and advice on particular technical issues; as well as evaluation of the results. In addition, in order to effectively evaluate the impact of the label on China's market, CLASP further provided assistance to CNIS to collect data on both the efficiency distribution and product volume distribution of refrigerators on the market. This short report summarizes the status of Standards and Labeling program, current enforcement and monitoring mechanism in China, and states the importance of international collaborations.

Zhou, Nan



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.

Priego-Capote, Feliciano; Scherl, Alexander; Muller, Markus; Waridel, Patrice; Lisacek, Frederique; Sanchez, Jean-Charles



High-performance liquid chromatographic separation of pairs of isotopic labeled (deuterium\\/protium) molecules  

Microsoft Academic Search

The separation of n-octane-d0 and n-octane-d18 has been accomplished using a liquid chromatographic technique; baseline separation was exhibited with the n-octane-d18 eluting first. Thus, the liquid chromatographic separation exhibits an inverse isotope effect just as has been observed for gas chromatographic separations. An inverse isotope effect was observed for isotopomer pairs even when they were not separated sufficiently to exhibit

Buchang Shi; Burtron H. Davis



LASER APPLICATIONS AND OTHER TOPICS IN QUANTUM ELECTRONICS: Efficient excitation of media for laser isotope separation  

Microsoft Academic Search

A theoretical investigation is made of the photoionisation of a three-level medium intended for laser isotope separation. The results show that the highest ionisation efficiency can be achieved by dividing the process into two stages: coherent population inversion and photoionisation. A study is made of the possibility of inversion of three-level systems for various detunings of the laser frequencies from

S. K. Borisov; M. A. Kuz'mina; V. A. Mishin




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.  

PubMed Central

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 15N (or 13C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-15N (or 13C) 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 15N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-15N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl[15N]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



Discovery of histone modification crosstalk networks by stable isotope labeling of amino acids in cell culture mass spectrometry (SILAC MS).  


In this paper we describe an approach that combines stable isotope labeling of amino acids in cells culture, high mass accuracy liquid chromatography tandem mass spectrometry and a novel data analysis approach to accurately determine relative peptide post-translational modification levels. This paper describes the application of this approach to the discovery of novel histone modification crosstalk networks in Saccharomyces cerevisiae. Yeast histone mutants were generated to mimic the presence/absence of 44 well-known modifications on core histones H2A, H2B, H3, and H4. In each mutant strain the relative change in H3 K79 methylation and H3 K56 acetylation were determined using stable isotope labeling of amino acids in cells culture. This approach showed relative changes in H3 K79 methylation and H3 K56 acetylation that are consistent with known histone crosstalk networks. More importantly, this study revealed additional histone modification sites that affect H3 K79 methylation and H3 K56 acetylation. PMID:23592332

Guan, Xiaoyan; Rastogi, Neha; Parthun, Mark R; Freitas, Michael A



Stable-Isotope Labeled Hydrophobic Hydrazide Reagents for the Relative Quantification of N-linked Glycans by Electrospray Ionization Mass Spectrometry  

PubMed Central

This study presents the development of stable-isotope labeled hydrophobic, hydrazide reagents for the relative quantification of N-linked glycans. The P2GPN ‘light’ (12C) and ‘heavy’ (13C6) pair are used to differentially label two N-linked glycan samples. The samples are combined 1:1, separated using HILIC, and then mass differentiated and quantified using mass spectrometry. These reagents have several benefits: 1) impart hydrophobic character to the glycans affording an increase in electrospray ionization efficiency and MS detection; 2) indistinguishable chromatographic, MS, and MS/MS performance of the ‘light’ and ‘heavy’ reagents affording relative quantification; and 3) analytical variability is significantly reduced due to the two samples being mixed together after sample preparation. Obtaining these analytical benefits only requires ~4 hours of sample preparation time. It is shown that these reagents are capable of quantifying changes in glycosylation in simple mixtures, and the analytical variability of the reagents in pooled plasma samples is shown to be less than ±30%. Additionally, the incorporation of an internal standard allows one to account for the difference in systematic error between the two samples due to the samples being processed in parallel and not mixed until after derivatization.

Walker, S. Hunter; Budhathoki-Uprety, Januka; Novak, Bruce M.; Muddiman, David C.



Innovative method for carbon dioxide determination in human postmortem cardiac gas samples using headspace-gas chromatography-mass spectrometry and stable labeled isotope as internal standard.  


A novel approach to measure carbon dioxide (CO2) in gaseous samples, based on a precise and accurate quantification by (13)CO2 internal standard generated in situ is presented. The main goal of this study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable in the routine determination of CO2. The main drawback of the GC methods discussed in the literature for CO2 measurement is the lack of a specific internal standard necessary to perform quantification. CO2 measurement is still quantified by external calibration without taking into account analytical problems which can often occur considering gaseous samples. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in situ an internal labeled standard gas ((13)CO2) on the basis of the stoichiometric formation of CO2 by the reaction of hydrochloric acid (HCl) with sodium hydrogen carbonate (NaH(13)CO3). This method allows a precise measurement of CO2 concentration and was validated on various human postmortem gas samples in order to study its efficiency. PMID:23746406

Varlet, V; Smith, F; de Froidmont, S; Dominguez, A; Rinaldi, A; Augsburger, M; Mangin, P; Grabherr, S



Efficient in vitro labeling of human prostate cancer cells with superparamagnetic iron oxide nanoparticles.  


The purpose of this study was to investigate the feasibility and optimization of protocols using superparamagnetic iron oxide (SPIO) nanoparticles to label human prostate cancer cell lines PC3 in vitro. The PC3 cells were labeled with different concentrations (28-252??g Fe/mL) of SPIO and increasing incubation time (6-24 hours), in the presence or absence of a transfection agent poly-l-lysine (PLL). The cell labeling efficiency was analyzed by Prussian blue stain method. The cellular viability was evaluated using trypan blue dye exclusion test. The signal intensity change of the labeled cells was assessed with magnetic resonance imaging (MRI). The results demonstrated that the iron oxide uptake by PC3 cells was dependent on dose and time. The PLL significantly increased the iron load of cells (p<0.01). A final concentration of SPIO nanoparticles of 42-126??g/mL with 12-24 hours incubation times could be sufficient to label PC3 cells for cellular MRI without impairment of cell viability. This technology may allow for further study into the mechanisms underlying prostate cancer progression as well as permit the real-time imaging of the effectiveness of cancer therapies in vivo. PMID:21812654

Jiang, Jun; Chen, Yaqing; Zhu, Yunkai; Yao, Xiaohong; Qi, Jun



Status of the Local Enforcement of Energy Efficiency Standards and Labeling Program in China  

SciTech Connect

As part of its commitment to promoting and improving the local enforcement of appliance energy efficiency standards and labeling, the China National Institute of Standardization (CNIS) launched the National and Local Enforcement of Energy Efficiency Standards and Labeling project on August 14, 2009. The project’s short-term goal is to expand the effort to improve enforcement of standards and labeling requirements to the entire country within three years, with a long-term goal of perfecting overall enforcement. For this project, Jiangsu, Shandong, Sichuan and Shanghai were selected as pilot locations. This report provides information on the local enforcement project’s recent background, activities and results as well as comparison to previous rounds of check-testing in 2006 and 2007. In addition, the report also offers evaluation on the achievement and weaknesses in the local enforcement scheme and recommendations. The results demonstrate both improvement and some backsliding. Enforcement schemes are in place in all target cities and applicable national standards and regulations were followed as the basis for local check testing. Check testing results show in general high labeling compliance across regions with 100% compliance for five products, including full compliance for all three products tested in Jiangsu province and two out of three products tested in Shandong province. Program results also identified key weaknesses in labeling compliance in Sichuan as well as in the efficiency standards compliance levels for small and medium three-phase asynchronous motors and self-ballasted fluorescent lamps. For example, compliance for the same product ranged from as low as 40% to 100% with mixed results for products that had been tested in previous rounds. For refrigerators, in particular, the efficiency standards compliance rate exhibited a wider range of 50% to 100%, and the average rate across all tested models also dropped from 96% in 2007 to 63%, possibly due to the implementation of newly strengthened efficiency standards in 2009. Areas for improvement include: Greater awareness at the local level to ensure that all manufacturers register their products with the label certification project and to minimize their resistance to inspections; improvement of the product sampling methodology to include representative testing of both large and small manufacturers and greater standardization of testing tools and procedures; and continued improvement in local enforcement efforts.

Zhou, Nan; Zheng, Nina; Fino-Chen, Cecilia; Fridley, David; Ning, Cao



Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells.  


The advent of the multiwavelength anomalous diffraction phasing method has significantly accelerated crystal structure determination and has become the norm in protein crystallography. This method allows researchers to take advantage of the anomalous signal from diverse atoms, but the dominant method for derivative preparation is selenomethionine substitution. Several generally applicable, high-efficiency labeling protocols have been developed for use in the bacterial, yeast, and baculovirus/insect cell expression systems but not for mammalian tissue culture. As a large number of proteins of biomedical importance can only be produced in yields sufficient for X-ray diffraction experiments in mammalian expression systems, it becomes all the more important to develop such protocols. We therefore evaluated several variables that play roles in determining incorporation levels and report here a simple protocol for selenomethionine modification of proteins in mammalian cells routinely yielding >90% labeling efficiency. PMID:16823037

Barton, William A; Tzvetkova-Robev, Dorothea; Erdjument-Bromage, Hediye; Tempst, Paul; Nikolov, Dimitar B



Potential bias and mitigations when using stable isotope labeled parent drug as internal standard for LC–MS\\/MS quantitation of metabolites  

Microsoft Academic Search

In recent years, increasing emphasis has been placed on quantitative characterization of drug metabolites for better insight into the correlation between metabolite exposure and toxicological observations or pharmacological efficacy. One common strategy for metabolite quantitation is to adopt the stable isotope labeled (STIL) parent drug as the internal standard in an isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS\\/MS) assay. In

Wenying Jian; Richard W. Edom; Yaodong Xu; Joseph Gallagher; Naidong Weng



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



Sulfur-34S stable isotope labeling of amino acids for quantification (SULAQ34) of proteomic changes in Pseudomonas fluorescens during naphthalene degradation.  


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 (13)C or (15)N labeled substrates might not be practical. Here, the incorporation of heavy sulfur isotopes is shown to be a useful alternative. We introduce (34)S 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



Efficient preparation and labeling of human induced pluripotent stem cells by nanotechnology  

PubMed Central

Efficient preparation and labeling of human induced pluripotent stem (iPS) cells is a great challenge in stem cell research and development. With the aim of investigating the feasibility of using nanotechnology to enhance the preparation efficiency of iPS cells and to label iPS cells for long-term tracing and imaging, in this paper, four transcription factor genes, ie, Oct4, Sox2, LIN28, and Nanog, and packaging plasmids such as PSPAX2 and PMD2.G were cotransfected into 293T cells using Generation 5.0 polyamidoamine dendrimer-modified magnetic nanoparticles (dMNPs) as a delivery system. The resultant supernatant liquids were incubated with human fibroblast cells at 37°C for 21 days, then the embryonic stem (ES) cell-like clones were screened, cultured, and identified. Finally, the prepared iPS cells were labeled with fluorescent magnetic nanoparticles (FMNPs). The results showed that dMNPs can efficiently deliver all vectors into 293T cells. The resultant lentiviruses’ titers were 10-fold more than those based on Lipofectamine™ 2000. Reverse transcription polymerase chain reaction analysis showed that four genes (Oct4, Sox2, LIN28, and Nanog) exhibited different expressions in iPS cells. Immunostaining analysis showed that specific surface markers of ES cells such as SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81 were positive in iPS cells, and the terotomas were formed in NOD-SCID mice that were implanted with iPS cells. Red fluorescent signals could be observed in iPS cells labeled with FMNPs by fluorescent microscopy, and the magnetic signals were detected in labeled iPS cells by magnetic resonance imaging. In conclusion, human iPS cells can be efficiently generated using polyamidoamine dMNPs and lentivirus and labeled with FMNPs for long-term observation and tracking, which has great potential application in the research and development of stem cells in the near future.

Ruan, Jing; Shen, Jie; Wang, Zheng; Ji, Jiajia; Song, Hua; Wang, Kan; Liu, Bin; Li, Jinhui; Cui, Daxiang



Measurement of atmospheric trace sulfur gases using combined gas chromatography - mass spectrometry and an isotopically labeled internal standard  

SciTech Connect

A method of combined mass spectrometry/gas chromatography based on an internal standard coupled with the concentration measurements is described for the determination of picogram quantities of certain sulfur gases. The lower level of detection was 4 ppT by volume at a signal-to-noise ratio of 2 for a 1 liter air sample with a precision of 1%. Signal ion overlap between the monitored mass channels was observed and an algorithm was derived to correct the isotopic ratio for this cross contribution. The algorithm defined a linear relationship between the detected isotopic signal ratio and the concentration of the measured compounds. The technique was applied to the determination of carbon disulfide and carbonyl sulfide at the Drexel University campus located in Philadelphia, PA. Two different sampling devices, a glass sampler and a carbosieve B solid adsorbent trap were investigated for collection of air samples, and high efficiency was demonstrated for both samplers. (BLM)

Shalaby, L.M.



Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters†  

PubMed Central

Deuterated styrene ([2H8]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [2H8]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [2H8]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.

Alexandrino, Maria; Knief, Claudia; Lipski, Andre



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.

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



PICquant: A Quantitative Platform to Measure Differential Peptide Abundance Using Dual-Isotopic Labeling with 12C6- and 13C6-Phenylisocyanate  

PubMed Central

We have developed a complete system for the isotopic labeling, fractionation, and automated quantification of differentially expressed peptides that significantly facilitates candidate biomarker discovery. We describe a new stable mass tagging reagent pair, 12C6- and 13C6-phenylisocyanate (PIC), that offers significant advantages over currently available tags. Peptides are labeled predominantly at their amino termini and exhibit elution profiles that are independent of label isotope. Importantly, PIC-labeled peptides have unique neutral-mass losses upon CID fragmentation that enable charge state and label isotope identification and, thereby, decouple the sequence identification from the quantification of candidate biomarkers. To exploit these properties, we have coupled peptide fractionation protocols with a Thermo LTQ-XL LC-MS2 data acquisition strategy and a suite of automated spectrum analysis software that identifies quantitative differences between labeled samples. This approach, dubbed the PICquant platform, is independent of protein sequence identification and excludes unlabeled peptides that otherwise confound biomarker discovery. Application of the PICquant platform to a set of complex clinical samples showed that the system allows rapid identification of peptides that are differentially expressed between control and patient groups.

Lyons, Charles E.; Victor, Ken G.; Moshnikov, Sergey A.; Bachmann, Lorin M.; Baras, Alexander S.; Dettmann, Kathleen M.; Cross, Janet V.; Templeton, Dennis J.



Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology  

Microsoft Academic Search

An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass\\u000a spectrometry (MS\\/MS) method was developed for differential protein expression profiling in complex cellular extracts. The\\u000a estrogen positive MCF-7 cell line, cultured in the presence of 17?-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ)

Jenny M. Armenta; Ina Hoeschele; Iulia M. Lazar



Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites.  


The formation of reactive metabolites from a number of compounds was studied in vitro using a mixture of non-labeled and stable isotope labeled glutathione (GSH) as a trapping agent. GSH was labeled by incorporating [1,2-(13)C(2),(15)N]glycine into the tripeptide to give an overall increase of 3 Da over the naturally occurring substance. Detection and characterization of reactive metabolites was greatly facilitated by using the data-dependent scanning features of the linear ion trap mass spectrometers to give complimentary and confirmatory data in a single analytical run. A comparison was made by analyzing the samples simultaneously on a triple-stage quadrupole mass spectrometer operated in the constant neutral loss mode. The compounds studied included 2-acetamidophenol, 3-acetamidophenol, 4-acetamidophenol (acetaminophen), and flufenamic acid. GSH adducts for each of these compounds produced a characteristic pattern of 'twin ions' separated by 3 Da in the mass spectral data. This greatly facilitated the detection and characterization of any GSH-related adducts present in the microsomal extracts. Furthermore, characterization of these adducts was greatly facilitated by the rapid scanning capability of linear ion trap instruments that provided full-scan, MS/MS and MS(3) data in one single analysis. This method of detecting and characterizing reactive metabolites generated in vitro was found to be far superior to any of the existing methods previously employed in this laboratory. The combination of two techniques, stable isotope labeled glutathione and linear ion traps, provided a very sensitive and specific method of identifying compounds capable of producing reactive metabolites in a discovery setting. The complimentary set of mass spectral data (including full-scan, MS/MS and MS(3) mass spectra), obtained rapidly in a single analysis with the linear ion trap instruments, greatly accelerated identification of metabolically bioactivated soft spots on the molecules. This in turn enabled chemists to rapidly design out the potential metabolic liability from the back-up compounds by making appropriate structural modifications. PMID:16261644

Mutlib, Abdul; Lam, Wing; Atherton, Jim; Chen, Hao; Galatsis, Paul; Stolle, Wayne



Using carbon isotope fractionation for an improved quantification of CH4 oxidation efficiency in Arctic peatlands  

NASA Astrophysics Data System (ADS)

Much research effort is focused on identifying global CH4 sources and sinks to estimate their current and potential strength in response to land-use change and global warming. Aerobic CH4 oxidation is regarded as the key process reducing the strength of CH4 emissions in wetlands, but is hitherto difficult to quantify. Recent studies quantify the efficiency of CH4 oxidation based on CH4 stable isotope signatures. The approach utilizes the fact that a significant isotope fractionation occurs when CH4 is oxidized. Moreover, it also considers isotope fractionation by diffusion. For field applications the 'open-system equation' is applied to determine the CH4 oxidation efficiency: fox = (?E - ?P)/ (?ox - ?trans) where fox is the fraction of CH4 oxidized; ?E is ?13C of emitted CH4; ?P is ?13C of produced CH4; ?ox is the isotopic fractionation factor of oxidation; ?trans is the isotopic fractionation factor of transport. We quantified CH4 oxidation in polygonal tundra soils of Russia's Lena River Delta analyzing depth profiles of CH4 concentrations and stable isotope signatures. Therefore, both fractionation factors ?ox and ?trans were determined for three polygon centers with differing water table positions and a polygon rim. While most previous studies on landfill cover soils have assumed a gas transport dominated by advection (?trans = 1), other CH4 transport mechanisms as diffusion have to be considered in peatlands and ?trans exceeds a value of 1. At our study we determined ?trans = 1.013 ± 0.003 for CH4 when diffusion is the predominant transport mechanism. Furthermore, results showed that ?ox differs widely between sites and horizons (?ox = 1.013 ± 0.012) and has to be determined for each case. The impact of both fractionation factors on the quantification of CH4 oxidation was estimated by considering both the potential diffusion rate at different water contents and potential oxidation rates. Calculations for a water saturated tundra soil indicated a CH4 oxidation efficiency of 88% in the upper horizon. Using carbon isotope fractionation improves the in situ quantification of CH4 oxidation in wetlands and thus the assessment of current and potential CH4 sources and sinks in these ecosystems.

Preuss, I.; Knoblauch, C.; Gebert, J.; Pfeiffer, E.-M.



A high efficiency device for the detection of radioactive xenon isotopes  

Microsoft Academic Search

A detector has been designed for the detection of sub-Becquerel amounts of the radioactive xenon isotopes 131mXe and 133Xe. High efficiency has been obtained by the condensation of the sample directly onto the surfaces of two closely spaced silicon surface-barrier detectors. The measurement of the 131mXe is based on the detection of the conversion electrons. The 133Xe measurement is obtained

G. P. Lamaze



Lewis Acid-Base, Molecular Modeling, and Isotopic Labeling in a Sophomore Inorganic Chemistry Laboratory  

ERIC Educational Resources Information Center

|An experiment to prepare a deuterium labeled adduct of a Lewis acid and Lewis base, to use computational methods allowing students to visualize the LUMO of Lewis acids, the HOMO of Lewis bases and the molecular orbitals of the adduct that is formed is developed. This allows students to see the interplay between calculated and experimental…

Nataro, Chip; Ferguson, Michelle A.; Bocage, Katherine M.; Hess, Brian J.; Ross, Vincent J.; Swarr, Daniel T.



Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya.  


The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC-MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study. PMID:19154813

Ocaña, Mireia Fernández; Fraser, Paul D; Patel, Raj K P; Halket, John M; Bramley, Peter M



Deciphering systemic wound responses of the pumpkin extrafascicular phloem by metabolomics and stable isotope-coded protein labeling.  


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, Jörg



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



Highly efficient cellular labeling of mesoporous nanoparticles in human mesenchymal stem cells: implication for stem cell tracking  

Microsoft Academic Search

Tracking the distribution of stem cells is crucial to their therapeutic use. However, the usage of current vectors in cellular labeling is restricted by their low internalizing efficiency. Here, we reported a cellular labeling approach with a novel vector composed of mesoporous silica nanoparticles (MSNs) conjugated with fluorescein isothiocyanate in human bone marrow mesenchymal stem cells and 3T3-L1 cells, and

Dong-Ming Huang; Yann Hung; Bor-Sheng Ko; Szu-Chun Hsu; Wei-Hsuan Chen; Chung-Liang Chien; Chih-Pin Tsai; Chieh-Ti Kuo; Ju-Chiun Kang; Chung-Shi Yang; Chung-Yuan Mou; Yao-Chang Chen



Lipids of symbiotic methane-oxidizing bacteria in peat moss studied using stable carbon isotopic labelling  

Microsoft Academic Search

Aerobic symbiotic methane-oxidizing bacteria (methanotrophs) in peat moss (Sphagnum spp.) play a vital role in the carbon cycle in peat bogs. They reduce methane emissions and provide CO2 to Sphagnum moss, resulting in effective in situ carbon recycling. To establish biomarkers for these methanotrophs, Sphagnum moss spp. were incubated with 13CH4 and analysed for the degree of label incorporation in

Julia F. van Winden; Nardy Kip; Gert-Jan Reichart; Mike S. M. Jetten; Jaap S. Sinninghe Damsté



Investigation of biosynthetic pathways to hydroxycoumarins during post-harvest physiological deterioration in Cassava roots by using stable isotope labelling.  


Cassava (Manihot esculenta Crantz) is an important starch-rich crop, but the storage roots only have a short shelf-life due to post-harvest physiological deterioration (PPD), which includes the over-production and polymerisation of hydroxycoumarins. Key aspects of coumarin secondary-metabolite biosynthesis remain unresolved. Here we exploit the accumulation of hydroxycoumarins to test alternative pathways for their biosynthesis. Using isotopically labelled intermediates (p-coumarate-2-(13)C, caffeate-2-(13)C, ferulate-2-(13)C, umbelliferone-2-(18)O and esculetin-2-(18)O), we show that the major biosynthetic pathway to scopoletin and its glucoside, scopolin, in cassava roots during PPD is through p-coumaric, caffeic and then ferulic acids. An alternate pathway through 2',4'-dihydroxycinnamate and umbelliferone leads to esculetin and esculin. We have used C(18)O(2)-carboxylate-labelled cinnamic and ferulic acids, and feeding experiments under an atmosphere of (18)O(2), to investigate the o-hydroxylation and cyclisation steps. We demonstrate that the major pathway is through o-hydroxylation and not via a proposed spirolactone-dienone intermediate. PMID:19035613

Bayoumi, Soad A L; Rowan, Michael G; Beeching, John R; Blagbrough, Ian S



Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters  

PubMed Central

Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.

Frias-Lopez, Jorge; Thompson, Anne; Waldbauer, Jacob; Chisholm, Sallie W



Probing AGXT2 enzyme activity in mouse tissue by applying stable isotope-labeled asymmetric dimethyl arginine as substrate.  


Asymmetric dimethylarginine (ADMA) is a metabolite of the amino acid L-arginine. It competitively inhibits the enzymatic production of the cell-signaling substance nitric oxide. Therefore, increased levels of ADMA are associated with a range of cardiovascular and other diseases. ADMA is biologically eliminated by direct renal excretion and hydrolysis by the enzyme DDAH. Recently, a further elimination pathway via the transamination by the enzyme AGXT2 to ?-keto-?-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) has come into the focus of biological research. In this work, we describe an assay for the AGXT2 activity in mouse liver and kidney tissue. It is based on the transformation of isotope-labeled ADMA-d(6) to DMGV-d(6). The quantification of the DMGV-d(6) produced by this reaction in tissue homogenate samples was accomplished by chromatographic separation on a porous graphitic carbon column and tandem mass spectrometric detection. DMGV-d(6) with the deuterium labels in different molecular positions was used as internal standard. The overall production rates of DMGV-d(6) in mice were 195.37?pmol/min/mg total protein in liver and 85.21?pmol/min/mg total protein in kidney tissue, with coefficients of variation of 6.31% and 11.25%, respectively. This method can be applied as a tool for the characterization of the ADMA elimination by the AGXT2 pathway. PMID:23280748

Martens-Lobenhoffer, Jens; Rodionov, Roman N; Bode-Böger, Stefanie M



Improved quantification of microbial CH4 oxidation efficiency in arctic wetland soils using carbon isotope fractionation  

NASA Astrophysics Data System (ADS)

Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the arctic will cause deeper permafrost thawing, followed by increased carbon mineralization and CH4 formation in water-saturated tundra soils, thus creating a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and ?13CH4 signatures were measured and the fractionation factors for the processes of oxidation (?ox) and diffusion (?diff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (such as landfill cover soils) have assumed a gas transport dominated by advection (?trans = 1). In tundra soils, however, diffusion is the main gas transport mechanism and diffusive stable isotope fractionation should be considered alongside oxidative fractionation. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an ?diff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was ?diff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that ?ox differs widely between sites and horizons (mean ?ox = 1.017 ± 0.009) and needs to be determined on a case by case basis. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged, organic-rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost-affected ecosystems and their potential strengths in response to global warming.

Preuss, I.; Knoblauch, C.; Gebert, J.; Pfeiffer, E.-M.



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



A Low-Storage-Consumption XML Labeling Method for Efficient Structural Information Extraction  

NASA Astrophysics Data System (ADS)

Recently, labeling methods to extract and reconstruct the structural information of XML data, which are important for many applications such as XPath query and keyword search, are becoming more attractive. To achieve efficient structural information extraction, in this paper we propose C-DO-VLEI code, a novel update-friendly bit-vector encoding scheme, based on register-length bit operations combining with the properties of Dewey Order numbers, which cannot be implemented in other relevant existing schemes such as ORDPATH. Meanwhile, the proposed method also achieves lower storage consumption because it does not require either prefix schema or any reserved codes for node insertion. We performed experiments to evaluate and compare the performance and storage consumption of the proposed method with those of the ORDPATH method. Experimental results show that the execution times for extracting depth information and parent node labels using the C-DO-VLEI code are about 25% and 15% less, respectively, and the average label size using the C-DO-VLEI code is about 24% smaller, comparing with ORDPATH.

Liang, Wenxin; Takahashi, Akihiro; Yokota, Haruo


Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications  

Microsoft Academic Search

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([15N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine\\/xanthine oxidase (100 nM min?1) and NADPH\\/calcium-calmodulin\\/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5?-dimethyl-1-pyrroline N-oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO–OOH), the superoxide adduct of EMPO (EMPO–OOH) does not spontaneously

Hao Zhang; Joy Joseph; Jeannette Vasquez-Vivar; Hakim Karoui; Cline Nsanzumuhire; Pavel Martásek; Paul Tordo; B Kalyanaraman



High efficiency Hall effect micro-biosensor platform for detection of magnetically labeled biomolecules.  


Detection of magnetically labeled biomolecules using micro-Hall biosensors is a promising method for monitoring biomolecular recognition processes. The measurement efficiency of standard systems is limited by the time taken for magnetic beads to reach the sensing area of the Hall devices. Here, micro-current lines were integrated with Hall effect structures to manipulate the position of magnetic beads via field gradients generated by localized currents flowing in the current lines. Beads were accumulated onto the sensor surface within seconds of passing currents through the current lines. Real-time detection of magnetic beads using current lines integrated with Hall biosensors was achieved. These results are promising in establishing Hall biosensor platforms as efficient and inexpensive means of monitoring biomolecular reactions for medical applications. PMID:17055242

Sandhu, Adarsh; Kumagai, Yoshimichi; Lapicki, Adam; Sakamoto, Satoshi; Abe, Masanori; Handa, Hiroshi



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.

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



High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies.  


The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure. PMID:17900926

Berkner, Hanna; Engelhorn, Julia; Liscombe, David K; Schweimer, Kristian; Wöhrl, Birgitta M; Facchini, Peter J; Rösch, Paul; Matecko, Irena



The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species  

PubMed Central

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri­glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly 13C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 ?l of plasma.

McLaren, David G.; He, Timothy; Wang, Sheng-Ping; Mendoza, Vivienne; Rosa, Raymond; Gagen, Karen; Bhat, Gowri; Herath, Kithsiri; Miller, Paul L.; Stribling, Sloan; Taggart, Andrew; Imbriglio, Jason; Liu, Jinqi; Chen, Dunlu; Pinto, Shirly; Balkovec, James M.; DeVita, Robert J.; Marsh, Donald J.; Castro-Perez, Jose M.; Strack, Alison; Johns, Douglas G.; Previs, Stephen F.; Hubbard, Brian K.; Roddy, Thomas P.



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.

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.  


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



Understanding cellular metabolism using stable isotopic labeling, metabolic flux analysis and kinetic modeling  

Microsoft Academic Search

There is focus on increasing scent production in flowers by metabolic engineering due to its broad impact in the perfume and horticulture industry. Metabolic engineering of flowers to enhance the scent production can be done in an efficient and targeted manner, if there is mechanistic information about the kinetics and regulation of the pathways involved in scent production. Thus a

Neelanjan Sengupta



Isolation of aminoacyl-tRNA and its labeling with stable-isotope tracers: Use in studies of human tissue protein synthesis  

SciTech Connect

The authors isolated aminoacyl-tRNA from human and rat tissues and measured, by GC/MS, its labeling in vivo by ({sup 15}N)-and ({sup 13}C)leucine. Tracer dilution artifacts seemed unlikely since, after infusion of L-(1-{sup 13}C, {sup 15}N)leucine into rats, (1) muscle leucyl-tRNA labeling exceeded tissue free leucine labeling, (2) values were largely unaffected by storing over 5 min at 22C, and (3) L-(2,4,5-methyl-{sup 13}C)leucine was not incorporated into leucyl-tRNA during homogenization. Leucyl-tRNA labeling in liver and muscle suggested charging from extra- and intracellular pools: e.g., after infusing L-(1-{sup 13}C, {sup 15}N)leucine, rat muscle tissue free leucine {sup 13}C labeling exceeded that by {sup 15}N and both were significantly lower than venous plasma indicating tracer dilution by transamination and by proteolysis; however, leucyl-tRNA labeling by either isotope was significantly above mixed tissue free leucing. Human placental leucyl-tRNA labeling (after predelivery tracer infusion) was 37% lower than maternal uterine vein labeling but not significantly different from placental free leucine or umbilical arterial leucine.

Watt, P.W.; Lindsay, Y.; Scrimgeour, C.M.; Chien, P.A.F.; Taylor, D.J.; Rennie, M.J. (Univ. of Dundee (England)); Gibson, J.N.A. (Univ. of Dundee (England) Univ. of Edinbergh (England))



Amyloid-beta isoform metabolism quantitation by stable isotope-labeled kinetics.  


Abundant evidence suggests a central role for the amyloid-beta (A?) peptide in Alzheimer's disease (AD) pathogenesis. Production and clearance of different A? isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify A? isoforms and quantitate them by the specific C-terminal peptides in order to investigate A? isoform physiology in the central nervous system. By using standards generated from in vitro metabolic labeling, the relative quantitation of four peptides representing total amount of A? (A?-Total), A?38, A?40, and A?42 were achieved both in cell culture and in human cerebrospinal fluid (CSF). Standard curves for each isoform demonstrated good sensitivity with very low limits of detection and high accuracy. Because the assay does not require antibody development for each A? isoform peptide, significant improvements in the throughput and accuracy of isoform quantitation were achieved. PMID:23714261

Mawuenyega, Kwasi G; Kasten, Tom; Sigurdson, Wendy; Bateman, Randall J



Magic angle sample spinning sup 13 C nuclear magnetic resonance of isotopically labeled bacteriorhodopsin  

SciTech Connect

Bacteriorhodopsin (bR), the light-driven proton pump protein from Halobacterium halobium, was biosynthetically labeled with (4-{sup 13}C)Asp. The incorporation yield was 48%. The magic angle sample spinning (MASS) {sup 13}C nuclear magnetic resonance (NMR) spectrum of this sample revealed six different peaks superimposed on a broad band of naturally abundant peptide-bond {sup 13}C. Two of the six carbonyl signals can be attributed to internal-protonated Asp carboxyl groups, one of which might be Asp115. An additional resonance at 110 ppm can be associated with the C-11 carbon of Trp, indicating an unusual biosynthetic pathway of this amino acid in Halobacterium halobium. Similar measurements performed on papain-treated purple membrane which lacks the C-terminal tail display two new intense signals at 178 and 178.9 ppm. If the same spectrum is taken without cross-polarization, these signals do not decrease or disappear. On the basis of their intensities and their chemical shifts, one can assign in addition to the C-terminal Asp four Asp residues facing the cytoplasmic phase. In native bR, at least two of these form a salt-bridge-like bond which also might include the C-terminal tail. These experiments not only provide data about the chemical environment of the Asp residues within the hydrophobic core of bacteriorhodopsin but also yield information about the interactions between surface components.

Engelhard, M.; Hess, B.; Emeis, D.; Metz, G.; Kreutz, W.; Siebert, F. (Max-Planck-Institut fuer Ernaehrungsphysiologie, Dortmund (Germany, F.R.))



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


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 protein detected from both lens types. The amount of protein extracted from contact lenses was affected by the lens material. Except for keratin-1 (0.83 ± 0.61 vs 0.77 ± 0.20, p = 0.81) or proline rich protein-4 (0.11 ± 0.04 vs 0.15 ± 0.12, p = 0.97), the amounts of lysozyme, lactoferrin, or lipocalin-1 extracted from balafilcon A lenses (12.9 ± 9.01, 0.84 ± 0.50 or 2.06 ± 1.6, respectively) were significantly higher than that extracted from senofilcon A lenses (0.88 ± 0.13, 0.50 ± 0.10 or 0.27 ± 0.23, respectively) (p < 0.05). The amount of protein extracted from contact lenses was dependent on both the individual wearer and the contact lens material. This may have implications for the development of clinical responses during lens wear for different people and with different types of contact lenses. The use of MRM-MS is a powerful analytical tool for the quantification of specific proteins from single contact lenses after wear. PMID:22784025

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



Isotopic labeling studies of interactions of nitric oxide and nitrous oxide with ultrathin oxynitride layers on silicon  

NASA Astrophysics Data System (ADS)

The interaction of nitric (NO) and nitrous (N2O) oxide with ultrathin (~1.5-3.5 nm) oxide and oxynitride films on silicon has been studied by performing high resolution depth profiling using medium energy ion scattering and isotopic labeling methods. We observe that, after NO annealing at 850 °C, both O and N incorporate near the SiO2/Si interface. There is no nitrogen and little newly incorporated oxygen observed at the surface, implying that NO diffuses through the oxide film and dissociates and reacts at the interface. For N2O annealing, atomic oxygen resulting from decomposition of the gas can replace oxygen atoms in both oxide and oxynitride films. This replacement is most important at the surface, but also, to a smaller extent, occurs in the middle of the film. For ultrathin oxynitride films, oxide growth during reoxidation is faster in N2O than in pure O2. Atomic oxygen also influences the nitrogen distribution, which moves further into the film and accumulate at the new interface. We discuss the roles of atomic oxygen and peroxyl bridging oxygen species in explaining the observed phenomena.

Lu, H. C.; Gusev, E. P.; Garfunkel, E.; Busch, B. W.; Gustafsson, T.; Sorsch, T. W.; Green, M. L.



Fourier Transform Infrared Spectroscopy andSite-Directed Isotope Labeling as a ProbeofLocalSecondary Structure intheTransmembrane DomainofPhospholamban  

Microsoft Academic Search

transform infrared (ATR-FTIR) spectroscopy alongwithsite-directed isotope labeling to probe thelocal structure ofhPLB.Thefrequency anddichroism oftheamideIand11bandsappearing at1658cm-1and1544 cm-1,respectively, showthatdehydrated andhydrated hPLBreconstituted intodimyristoylphosphatidylcholine bilayer membranes ispredominantly a-helical andhasa nettransmembrane orientation. Specific local secondary structure ofhPLB was probedbyincorporating 13Cattwopositions intheprotein backbone. A small bandseen near1614cm-1isassigned totheamideImodeofthe13C-labeled amidecarbonyl group(s). Thefrequency anddichroism ofthisbandindicate that residues 39and46area-helical, with an axial orientation thatisapproximately 300relative tothemembranenormal. Upon exposure to

Cheryl F. C. Ludlam; Isaiah T. Arkin; Xiao-Mei Liu; Parshuram Rath


Residue-Specific Structural Kinetics of Proteins through the Union of Isotope Labeling, Mid-IR Pulse Shaping, and Coherent 2D IR Spectroscopy  

PubMed Central

We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for 13C=18O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide form type 2 diabetes.

Middleton, Chris T.; Woys, Ann Marie; Mukherjee, Sudipta S.; Zanni, Martin T.



Synthesis of isotopically-labeled graphite films by cold-wall chemical vapor deposition and electronic properties of graphene obtained from such films  

Microsoft Academic Search

We report the synthesis of isotopically-labeled graphite films on nickel substrates by using cold-wall chemical vapor deposition\\u000a (CVD). During the synthesis, carbon from 12C- and 13C-methane was deposited on, and dissolved in, a nickel foil at high temperature, and a uniform graphite film was segregated\\u000a from the nickel surface by cooling the sample to room temperature. Scanning and transmission electron

Weiwei Cai; Richard D. Piner; Yanwu Zhu; Xuesong Li; Zhenbing Tan; Herman Carlo Floresca; Changli Yang; Li Lu; M. J. Kim; Rodney S. Ruoff



Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line  

SciTech Connect

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.

Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.; Guillon, G.; Homburger, V.



Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment  

Microsoft Academic Search

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging.\\u000a On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications\\u000a frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive.\\u000a One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is

Ying Fan; Lichi Shi; Vladimir Ladizhansky; Leonid S. Brown



New untargeted metabolic profiling combining mass spectrometry and isotopic labeling: application on Aspergillus fumigatus grown on wheat.  


Characterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus , an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MS(n)) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC-HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins. PMID:23901908

Cano, Patricia M; Jamin, Emilien L; Tadrist, Souria; Bourdaud'hui, Pascal; Péan, Michel; Debrauwer, Laurent; Oswald, Isabelle P; Delaforge, Marcel; Puel, Olivier



Pathway of oxygen incorporation from O2 in TiO2 photocatalytic hydroxylation of aromatics: oxygen isotope labeling studies.  


The hydroxylation process is the primary, and even the rate-determining step of the photocatalytic degradation of aromatic compounds. To make clear the hydroxylation pathway of aromatics, the TiO(2) photocatalytic hydroxylation of several model substrates, such as benzoic acid, benzene, nitrobenzene, and benzonitrile, has been studied by an oxygen-isotope-labeling method, which can definitively assign the origin of the O atoms (from oxidant O(2) or solvent H(2)O) in the hydroxyl groups of the hydroxylated products. It is found that the oxygen source of the hydroxylated products depends markedly on the reaction conditions. The percentage of the products with O(2)-derived hydroxyl O atoms increases with the irradiation time, while it decreases with the increase of substrate concentration. More intriguingly, when photogenerated valence-band holes (h(vb)(+)) are removed, nearly all the O atoms (>97?%) in the hydroxyl groups of the hydroxylated products of benzoic acid come from O(2), whereas the scavenging of conduction-band electrons (e(cb)(-)) makes almost all the hydroxyl O atoms (>95?%) originate from solvent H(2)O. In the photocatalytic oxidation system with benzoic acid and benzene coexisting in the same dispersion, the percentage of O(2)-derived hydroxyl O atoms in the hydroxylated products of strongly adsorbed benzoic acid (ca. 30?%) is much less than in that of weakly adsorbed benzene (phenol) (>60?%). Such dependences provide unique clues to uncover the photocatalytic hydroxylation pathway. Our experiments show that the main O(2)-incorporation pathway involves the reduction of O(2) by e(cb)(-) and the subsequent formation of free (?)OH via H(2)O(2), which was usually overlooked in the past photocatalytic studies. Moreover, in the hydroxylation initiated by h(vb)(+), unlike the conventional mechanism, the O atom in O(2) cannot incorporate into the product through the direct coupling between molecular O(2) and the substrate-based radicals. PMID:22266774

Li, Yue; Wen, Bo; Yu, Cailan; Chen, Chuncheng; Ji, Hongwei; Ma, Wanhong; Zhao, Jincai



Isotopic distributions.  


Isotopic information determined by mass spectrometry can be used in a wide variety of applications. Broadly speaking these could be classified as "passive" applications, meaning that they use naturally occurring isotopic information, and "active" applications, meaning that the isotopic distributions are manipulated in some way. The classic passive application is the determination of chemical composition by comparing observed isotopic patterns of molecules to theoretically calculated isotopic patterns. Active applications include isotope exchange experiments of a variety of types, as well as isotope labeling in tracing studies and to provide references for quantitation. Regardless of the type of application considered, the problem of theoretical calculation of isotopic patterns almost invariably arises. This paper reviews a number of application examples and computational approaches for isotopic studies in mass spectrometry. PMID:23666722

Rockwood, Alan L; Palmblad, Magnus



Methyl-coenzyme m reductase from methanogenic archaea: isotope effects on label exchange and ethane formation with the homologous substrate ethyl-coenzyme m.  


Ethyl-coenzyme M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a homologous substrate for the enzyme methyl-coenzyme M reductase (MCR) resulting in the product ethane instead of methane. The catalytic reaction proceeds via an intermediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-limiting step, many cycles of intermediate formation and reconversion to substrate occur before a substantial amount of ethane is released. In deuterated buffer, the intermediate becomes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables intermediate formation to be detected. Here, we present a comprehensive analysis of this pre-equilibrium. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the time course of each isotopologue was followed by NMR spectroscopy. A kinetic simulation including kinetic isotope effects allowed determination of the primary and ?- and ?-secondary isotope effects for intermediate formation and for the C-H/C-D bond activation in the ethane-containing intermediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller , S. ; Goenrich , M. ; Thauer , R. K. ; Jaun , B. J. Am. Chem. Soc. 2013 , 135 , DOI: 10.1021/ja406485z 10.1021/ja406485z ) and thus imply the same catalytic mechanism for both substrates. The experiment by Floss and co-workers, demonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is compatible with the observed rapid isotope exchange if the isotope effects measured here are taken into account. PMID:24003767

Scheller, Silvan; Goenrich, Meike; Thauer, Rudolf K; Jaun, Bernhard



Energy-Efficiency Labels and Standards: A Guidebook forAppliances, Equipment, and Lighting - 2nd Edition  

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 several other organizations identified on the cover of this guidebook 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 second edition of the guidebook was prepared over the course of the past year, four years after the preparation of the first edition, with a significant contribution from the authors and reviewers mentioned previously. Their diligent participation helps maintain this book as the international guidance tool it has become. The lead authors would like to thank the members of the Communications Office of the Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory for their support in the development, production, and distribution of the guidebook. 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 sponsor 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 may be downloaded to be used intact or piecemeal for whatever beneficial purposes readers may conceive.

Wiel, Stephen; McMahon, James E.



High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy  

PubMed Central

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.

Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.



Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry  

NASA Astrophysics Data System (ADS)

The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by 15 N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

Pettelkau, Jens; Thondorf, Iris; Theisgen, Stephan; Lilie, Hauke; Schröder, Thomas; Arlt, Christian; Ihling, Christian H.; Sinz, Andrea



GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling.  


The established GC-EI-TOF-MS method for the profiling of soluble polar metabolites from plant tissue was employed for the kinetic metabolic phenotyping of higher plants. Approximately 100 typical GC-EI-MS mass fragments of trimethylsilylated and methoxyaminated metabolite derivatives were structurally interpreted for mass isotopomer analysis, thus enabling the kinetic study of identified metabolites as well as the so-called functional group monitoring of yet non-identified metabolites. The monitoring of isotope dilution after (13)CO(2) labelling was optimized using Arabidopsis thaliana Col-0 or Oryza sativa IR57111 plants, which were maximally labelled with (13)C. Carbon isotope dilution was evaluated for short (2h) and long-term (3 days) kinetic measurements of metabolite pools in root and shoots. Both approaches were shown to enable the characterization of metabolite specific partitioning processes and kinetics. Simplifying data reduction schemes comprising calculation of (13)C-enrichment from mass isotopomer distributions and of initial (13)C-dilution rates were employed. Metabolites exhibited a highly diverse range of metabolite and organ specific half-life of (13)C-label in their respective pools ((13)C-half-life). This observation implied the setting of metabolite specific periods for optimal kinetic monitoring. A current experimental design for the kinetic metabolic phenotyping of higher plants is proposed. PMID:17475294

Huege, Jan; Sulpice, Ronan; Gibon, Yves; Lisec, Jan; Koehl, Karin; Kopka, Joachim



Stable isotopes as ecological tracers: an efficient method for assessing the contribution of multiple sources to mixtures  

NASA Astrophysics Data System (ADS)

Stable isotopes are increasingly being used as tracers of ecological processes potentially providing relevant information to environmental management issues. An application of the methodology consists in relating the stable isotopic composition of a sample mixture to that of sources. The number of stable isotopes, however, is usually lower than that of potential sources existing in an ecosystem, which creates mathematical difficulties in correctly tracing sources. We discuss a linear programming model which efficiently derives information on the contribution of sources to mixtures for any number of stable isotopes and any number of sources by addressing multiple sources simultaneously. The model identifies which sources are present in all, present in a subset of the samples or absent from all samples simultaneously and calculates minimum and maximum values of each source in the mixtures. We illustrate the model using a data set consisting on the isotopic signatures of different plant sources ingested by primary consumers in tropical riverine habitat in Asia. The model discussed may contribute to extend the scope of stable isotopes methodology to a range of new problems dealing with multiple sources and multiple tracers. For instance, in food web studies, if particular organic matter sources disappear or decrease in availability (e.g. climate change scenarios) the model allows simulation of alternative diets of the consumers providing potentially relevant information for managers and decision makers.

Bugalho, M. N.; Barcia, P.; Caldeira, M. C.; Cerdeira, J. O.



Stable isotopes as ecological tracers: an efficient method for assessing the contribution of multiple sources to mixtures  

NASA Astrophysics Data System (ADS)

Stable isotopes are increasingly being used as tracers of ecological processes potentially providing relevant information to environmental management issues. An application of the methodology consists in relating the stable isotopic composition of a sample mixture to that of sources. The number of stable isotopes, however, is usually lower than that of potential sources existing in an ecosystem, which creates mathematical difficulties in correctly tracing sources. We discuss a linear programming model which efficiently derives information on the contribution of sources to mixtures for any number of stable isotopes and any number of sources by addressing multiple sources simultaneously. The model identifies which sources are present in all, present in a subset of the samples or absent from all samples simultaneously and calculates minimum and maximum values of each source in the mixtures. We illustrate the model using a data set consisting of the isotopic signatures of different plant sources ingested by primary consumers in tropical riverine habitat in Asia. The model discussed may contribute to extend the scope of stable isotopes methodology to a range of new problems dealing with multiple sources and multiple tracers. For instance, in food web studies, if particular organic matter sources disappear or decrease in availability (e.g. climate change scenarios) the model allows simulation of alternative diets of the consumers providing potentially relevant information for managers and decision makers.

Bugalho, M. N.; Barcia, P.; Caldeira, M. C.; Cerdeira, J. O.



Efficient Microwave-Assisted Synthesis of Human Islet Amyloid Polypeptide Designed to Facilitate the Specific Incorporation of Labeled Amino Acids  

PubMed Central

A cost-efficient, time-reducing solid-phase synthesis of the amyloidogenic, 37 residue islet amyloid polypeptide (IAPP) is developed using two pseudoprolines (highlighted blue in sequence) in combination with microwave technology. A yield twice that obtained with conventional syntheses is realized. The utility of this protocol is demonstrated by the synthesis of a 13C18O-labeled Ser-20 IAPP variant, a prohibitively expensive and chemically challenging site to label via other protocols. TEM analysis shows the peptide forms normal amyloid.

Marek, Peter; Woys, Ann Marie; Sutton, Kelvin; Zanni, Martin T.; Raleigh, Daniel P.



Highly efficient and isotope selective photo-ionization of barium atoms using diode laser and LED light.  


We demonstrated a simple method to photo-ionize barium atoms using 791 nm diode laser together with 310 nm UV LED. It solved the bottle-neck problem of previous method using 791 nm diode laser and 337 nm N(2) laser, whose ionization rate was limited by the repetition rate of N(2) laser. Compared with previous method, it has advantages of high efficiency together with simple and cheap setups. By tuning the frequency of 791 nm laser to be resonant with the desired isotope, isotope selective photo-ionization has been realized. PMID:21935008

Wang, B; Zhang, J W; Gao, C; Wang, L J



Active parts for CH 4 decomposition and electrochemical oxidation at metal\\/oxide interfaces by isotope labeling-secondary ion mass spectrometry  

Microsoft Academic Search

Active parts for CH4 decomposition and electrochemical oxidation of reformed gases were investigated at the well defined Ni-mesh\\/oxide interfaces in ?m level. The effective reaction areas were determined by isotope labeling technique under the mixture of CH4, D2O, and 18O2 at 1073 K. A deposition of carbon preferentially occurred on the Ni-mesh surface on Y2O3-stabilized ZrO2 (YSZ) and Sm2O3-doped CeO2

Teruhisa Horita; Haruo Kishimoto; Katsuhiko Yamaji; Natsuko Sakai; Yueping Xiong; Manuel E. Brito; Harumi Yokokawa; Muneaki Rai; Koji Amezawa; Yoshiharu Uchimoto



NIR mega-Stokes fluorophores for bioorthogonal labeling and energy transfer systems--an efficient quencher for daunomycin.  


A set of new azide- and alkyne-bearing lepidinium-based fluorophores were synthesized for bioorthogonal labeling schemes. These fluorescent dyes all show large Stokes-shifts with emission maxima in the near-infrared (NIR) region of the electromagnetic spectrum. The applicability of these dyes in the construction of energy-transfer systems was tested using one of these new fluorescent tags and daunomycin (Dau), an anticancer drug with fluorescent features. These daunomycin conjugates are the very first examples of fluorescently modulated constructs of this anticancer agent. The dually labeled architectures proved that the applied fluorescent dye can be utilized as an efficient quencher for daunomycin. Enzymatic cleavage of a dually labeled enzyme substrate resulted in full recovery of the fluorescence of daunomycin. Such fluorescently modulated Dau conjugates can provide useful information for the mechanism of action of Dau-regulated cell death processes. PMID:23225500

Cserép, Gergely B; Enyedi, Kata N; Demeter, Attila; Mez?, Gábor; Kele, Péter



Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: Studies in vitro, in animals, and in humans  

Microsoft Academic Search

A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering (6,6-2H2)Glc or (U-13C)Glc, isolat- ing genomic DNA, hydrolyzing enzymatically to free deoxyri- bonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracel- lular




Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex.  

PubMed Central

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.

Kellenbach, E R; Remerowski, M L; Eib, D; Boelens, R; van der Marel, G A; van den Elst, H; van Boom, J H; Kaptein, R



BODIPY-labeled DC-SIGN-targeting glycodendrons efficiently internalize and route to lysosomes in human dendritic cells.  


Glycodendrons bearing nine copies of mannoses or fucoses have been prepared by an efficient convergent strategy based on Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC). These glycodendrons present a well-defined structure and have an adequate size and shape to interact efficiently with the C-type lectin DC-SIGN. We have selected a BODIPY derivative to label these glycodendrons due to its interesting physical and chemical properties as chromophore. These BODIPY-labeled glycodendrons were internalized into dendritic cells by mean of DC-SIGN. The internalized mannosylated and fucosylated dendrons are colocalized with LAMP1, which suggests routing to lysosomes. The interaction of these glycodendrons with DC-SIGN at the surface of dendritic cells did not induce maturation of the cells. Signaling analysis by checking different cytokines indicated also the lack of induction the expression of inflammatory and noninflammatory cytokines by these second generation glycodendrons. PMID:22920925

Ribeiro-Viana, Renato; García-Vallejo, Juan J; Collado, Daniel; Pérez-Inestrosa, Ezequiel; Bloem, Karien; van Kooyk, Yvette; Rojo, Javier



Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI  

Microsoft Academic Search

reverse heparin anticoagulation but also used ex vivo as a cationic transfection agent. After labeling of human mesenchy- mal stem cells (MSCs) and hematopoietic (CD34) stem cells and other mammalian cells with ferumoxides-protamine sulfate complexes (FE-Pro), cellular toxicity, func- tional capacity, and quantitative cellular iron incorporation were determined. FE- Pro-labeled cells demonstrated no short- or long-term toxicity, changes in differen-

Ali S. Arbab; Gene T. Yocum; Heather Kalish; Elaine K. Jordan; Stasia A. Anderson; Aarif Y. Khakoo; Elizabeth J. Read; Joseph A. Frank



A simple and efficient algorithm for high-quality line labeling  

Microsoft Academic Search

The interest in algorithms that automatically place labels on maps, graphs, or diagrams has\\u000aincreased with the advance in type-setting technology and the amount of information to be visualized.\\u000aHowever, though manually labeling a map is estimated to take fifty percent of total map\\u000aproduction time (Morrison, 1980), most geographic information systems (GIS) offer only very basic\\u000alabel-placement features. In

A. Wolff; Lars Knipping; Marc van Kreveld; Tycho Strijk; Pankaj K. Agarwal



Isotopic composition of H2 from wood burning: Dependency on combustion efficiency, moisture content, and ?D of local precipitation  

NASA Astrophysics Data System (ADS)

Differences in isotopic composition between the various sources of H2 are large, but only a few measurements have been carried out to constrain them. Two conflicting values have been published for H2 from biomass burning. Both rely on the assumption that the isotopic composition of H2 should scale with the isotopic composition of the precipitation at the location where the biomass grew. Here we test this hypothesis using 18 wood samples collected from various locations around the globe. The sample locations cover a range of ?D content of H2 in precipitation, from below -120‰ in Siberia and Canada to -15‰ in Zimbabwe. The results confirm the predicted dependence of the H2 isotopic composition on the precipitation in the sampling region. The water content itself is found to at most slightly affect the results. Furthermore, ?D of H2 depends strongly on combustion efficiency. Thus, the isotopic composition of H2 from biomass burning shows a strong variability around the globe and between different stages of a fire. It is suggested that, rather than a global bulk number, global models that attempt to reproduce the spatial and temporal distribution of ?D in H2 should incorporate explicitly the variability of ?D(H2) from biomass burning on ?D in precipitation.

RöCkmann, Thomas; Gómez ÁLvarez, Catalina X.; Walter, Sylvia; van der Veen, Carina; Wollny, Adam G.; Gunthe, Sachin S.; Helas, Günther; PöSchl, Ulrich; Keppler, Frank; Greule, Markus; Brand, Willi A.



Dual isotope labeling: Conjugation of (32)P-oligonucleotides with (18)F-aryltrifluoroborate via copper(I) catalyzed cycloaddition.  


A one-pot-two-step labeling of an oligonucleotide with an (18)F-ArBF3(-)(aryltrifluoroborate) radioprosthetic is reported herein. In order to characterize labeling in terms of radiochemistry, phosphorus-32 was also introduced to the 5'-terminus of the oligonucleotide via enzymatic phosphorylation. A pendant azide group was subsequently conjugated to the 5'-phosphate of the oligonucleotide. Copper(I) catalyzed [2+3] cycloaddition was undertaken to conjugate an alkyne-bearing(18)F-ArBF3(-) to the oligonucleotide. Following polyacrylamide gel electrophoresis, this doubly-labeled bioconjugate exhibited decay properties of both the phosphorus-32 and fluorine-18, that were confirmed by autoradiography at selected lengths of time, which in turn provided concrete evidence of successful conjugation. These results are corroborated by HPLC analysis of the labeled material. Taken together this work demonstrates viable use of (18)F-ArBF3(-) prosthetics for labeling oligonucleotides for use in PET imaging. PMID:24144852

Li, Ying; Schaffer, Paul; Perrin, David M



Validation of methane measurement using headspace-GC-MS and quantification by a stable isotope-labeled internal standard generated in situ.  


A previous study has shown the possibility to identify methane (CH4 ) using headspace-GC-MS and quantify it with a stable isotope as internal standard. The main drawback of the GC-MS methods discussed in literature for CH4 measurement is the absence of a specific internal standard necessary to perform quantification. However, it becomes essential to develop a safer method to limit the manipulation of gaseous CH4 and to precisely control the injected amount of gas for spiking and calibration by comparison with external calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate a labeled gas as an internal standard in a vial on the basis of the formation of CH4 by the reaction of Grignard reagent methylmagnesium chloride with deuterated water. This method allows precise measurement of CH4 concentrations in gaseous sample as well as in a solid or a liquid sample after a thermodesorption step in a headspace vial. A full accuracy profile validation of this method is then presented. PMID:23585415

Varlet, Vincent; Smith, Fiona; Augsburger, Marc



NTFD--a stand-alone application for the non-targeted detection of stable isotope-labeled compounds in GC/MS data  

PubMed Central

Summary: Most current stable isotope-based methodologies are targeted and focus only on the well-described aspects of metabolic networks. Here, we present NTFD (non-targeted tracer fate detection), a software for the non-targeted analysis of all detectable compounds derived from a stable isotope-labeled tracer present in a GC/MS dataset. In contrast to traditional metabolic flux analysis approaches, NTFD does not depend on any a priori knowledge or library information. To obtain dynamic information on metabolic pathway activity, NTFD determines mass isotopomer distributions for all detected and labeled compounds. These data provide information on relative fluxes in a metabolic network. The graphical user interface allows users to import GC/MS data in netCDF format and export all information into a tab-separated format. Availability: NTFD is C++- and Qt4-based, and it is freely available under an open-source license. Pre-compiled packages for the installation on Debian- and Redhat-based Linux distributions, as well as Windows operating systems, along with example data, are provided for download at Contact:

Hiller, Karsten; Wegner, Andre; Weindl, Daniel; Cordes, Thekla; Metallo, Christian M.; Kelleher, Joanne K.; Stephanopoulos, Gregory



High-resolution direct infusion-based mass spectrometry in combination with whole 13C metabolome isotope labeling allows unambiguous assignment of chemical sum formulas.  


A new strategy for direct infusion-based metabolite analysis employing a combination of high-resolution mass spectrometry and (13)C-isotope labeling of entire metabolomes is described. Differentially isotope labeled metabolite extracts from otherwise identically grown reference plants were prepared and infused into a Fourier transform ion cyclotron resonance mass spectrometer. The derived accurate mass lists from each extract were searched, using an in-house-developed database search tool, against a number of comprehensive metabolite databases. Comparison of the retrieved chemical formulas from both, the (12)C and (13)C samples, leads to two major advantages compared to nonisotope-based metabolite fingerprinting: first, removal of background contaminations from the result list, due to the (12)C/(13)C peak pairing principle and therefore positive identification of compounds of true biological origin; second, elimination of ambiguity in chemical formula assignment due to the same principle, leading to the clear association of one measured mass to only one chemical formula. Applying this combination of strategies to metabolite extracts of the model plant Arabidopsis thaliana therefore resulted in the reproducible identification of more than 1000 unambiguous chemical sum formulas of biological origin of which more than 80% have not been associated to Arabidopsis before. PMID:19072260

Giavalisco, Patrick; Hummel, Jan; Lisec, Jan; Inostroza, Alvaro Cuadros; Catchpole, Gareth; Willmitzer, Lothar



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.



Factors affecting the efficiency of immunogold labelling of plant virus antigens in thin sections.  


Sections of pellets of six purified plant viruses with three different morphologies were used to examine different technical aspects of the immunogold labelling (IGL) technique. The results showed that fixation by glutaraldehyde alone was better than with osmium tetroxide post-fixation, and that Decon 75 was the best of the pretreatments tried. The study showed that different virus homologous antisera gave different results in IGL tests, and that longer incubation times with both antiserum and gold probe gave higher label densities without any increase in background label. Also, cross-absorption of the virus antisera with healthy host protein before use gave cleaner backgrounds and thus higher specificity. The work also examined the relationship between label density and amounts of visible virus. There was no correlation between the numbers of virus particles seen in sections and the numbers of gold particles; moreover, there was no apparent relationship between label density and the orientation or distribution of the virus particles in the section. The role of the embedding resin and its polymerisation temperature are also discussed. PMID:7714038

Roberts, I M



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.



Use of an oral/intravenous dual-label stable-isotope protocol to determine folic acid bioavailability from fortified cereal grain foods in women.  


Folic acid fortification, mandatory in the United States, is currently being considered by the UK. The hypothesis that the matrix of some cereal-product vehicles may result in low fortificant bioavailability was tested using a dual oral/intravenous (i.v.) isotopic-label approach, which was evaluated concurrently. Fifteen women received 225 microg oral folate (capsules, fortified white bread and fortified branflakes), mainly as folic acid labeled with (13)C on 6 carbons of the benzoyl ring ((13)C(6)-PteGlu), followed by i.v. injection of 100 microg folic acid labeled with (2)H on 4 hydrogens of the glutamic acid group ((2)H(4)-PteGlu). The urinary excretion ratio (UER) in intact folate of the percentage of labeled oral dose excreted divided by the percentage of i.v. dose excreted was used as the primary index of absorption. The geometric mean (95% confidence interval) UER for folic acid capsules was 3.68 (1.90, 7.14) at 24 h and 2.18 (1.24, 3.83) at 48 h. Because these were significantly in excess of 1.0, indicative of 100% absorption of the oral dose, it was concluded that oral and i.v. labeled folic acid are handled differently by the body and that "absolute" absorption cannot be calculated. Compared with the 48-h UER for folic acid capsules, the "relative" 48-h UER for white bread and branflakes was 0.71 and 0.37, respectively, indicating that some cereal-based vehicles may inhibit absorption of fortificant. However, even the validity of this "relative" approach is questioned. PMID:11983817

Finglas, Paul M; Witthöft, Cornelia M; Vahteristo, Liisa; Wright, Anthony J A; Southon, Susan; Mellon, Fred A; Ridge, Brian; Maunder, Peter



Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli  

PubMed Central

Background Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. Results In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. Conclusions The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.



Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes.  


Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism. PMID:15272882

Roessner-Tunali, Ute; Liu, JunLi; Leisse, Andrea; Balbo, Ilse; Perez-Melis, Alicia; Willmitzer, Lothar; Fernie, Alisdair R



Fast and efficient DNA crosslinking and multiple orthogonal labelling by copper-free click chemistry.  


Two new dibenzocyclooctyne-thymidine monomers were incorporated into oligonucleotides and crosslinked to azide-labelled complementary strands across the DNA grooves. Equivalent reactions were successful using a (bicyclo[6.1.0]nonyne) alkyne. Oligonucleotides containing internal cyclooctyne and amino groups were simultaneously reacted with azides and NHS esters of different fluorescent dyes to produce functional genetic probes. PMID:22892959

Shelbourne, Montserrat; Brown, Tom; El-Sagheer, Afaf H; Brown, Tom



Production of stable-isotope-labeled bovine heme and its use to measure heme-iron absorption in children  

Technology Transfer Automated Retrieval System (TEKTRAN)

BACKGROUND: The use of stable isotopes has provided valuable insights into iron absorption in humans, but the data have been limited to nonheme iron. OBJECTIVE: Our objectives were to produce heme iron enriched in (58)Fe and to use it to study the absorption of heme iron and the effect of iron and ...


Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding.  


The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering. PMID:2464371

Tüchsen, E; Hansen, P E



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



Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.  

PubMed Central

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure. Images Fig. 1

Xu, J; Lapham, J; Crothers, D M



Does an energy efficiency label alter consumers' purchasing decisions? A latent class approach based on a stated choice experiment in Shanghai.  


In this paper we conducted a hypothetical choice experiment in Shanghai, China, to examine whether the China Energy Efficiency Label influences consumers' choices of air conditioners and refrigerators. A latent class approach was applied to observe both heterogeneities among the respondents and product brands. Our results suggested that consumers in Shanghai were well aware of the China Energy Efficiency Label and tended to pay more attention to products with such labels. In addition, air conditioners and refrigerators affixed with a hypothetical label that indicates saving in electricity bills compared with a standard model received significant preferences, which suggested that the more information manufacturers provide, the more their products would be preferred by consumers. Finally, weighted by class probability, the willingness to pay values for more energy efficient refrigerators were higher than those for more energy efficient air conditioners, implying that Shanghai consumers have greater incentive to pay more for appliances they use more frequently. PMID:19595499

Shen, Junyi; Saijo, Tatsuyoshi



Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry  

Microsoft Academic Search

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS\\/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information

Hui Zhang; Xiao-jun Li; Daniel B Martin; Ruedi Aebersold



Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL37 in Escherichia coli for NMR studies  

Microsoft Academic Search

Antimicrobial peptide LL-37 plays an important role in human body’s first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the incorporation of 15N and other isotopes into the polypeptide for nuclear magnetic resonance

Yifeng Li; Xia Li; Guangshun Wang



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.  


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 (13)C?(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies 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 line widths 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) line width. Narrower line widths 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



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))



A quantitative proteomic analysis of lung epithelial (A549) cells infected with 2009 pandemic influenza A virus using stable isotope labelling with amino acids in cell culture.  


Influenza A virus is one of the world's major uncontrolled pathogens, causing seasonal epidemics as well as global pandemics. This was evidenced by the recent emergence and now prevalence of the 2009 swine origin pandemic H1N1 influenza A virus. In this study, quantitative proteomics using stable isotope labelling with amino acids in cell culture was used to investigate the changes in the host cell proteome in cells infected with pandemic H1N1 influenza A virus. The study was conducted in A549 cells that retain properties similar to alveolar cells. Several global pathways were affected, including cell cycle regulation and lipid metabolism, and these could be correlated with recent microarray analyses of cells infected with influenza A virus. Taken together, both quantitative proteomics and transcriptomic approaches can be used to identify potential cellular proteins whose functions in the virus life cycle could be targeted for chemotherapeutic intervention. PMID:22585751

Dove, Brian K; Surtees, Rebecca; Bean, Thomas J H; Munday, Diane; Wise, Helen M; Digard, Paul; Carroll, Miles W; Ajuh, Paul; Barr, John N; Hiscox, Julian A



Efficient estimators for quantum instanton evaluation of the kinetic isotope effects: Application to the intramolecular hydrogen transfer in pentadiene  

NASA Astrophysics Data System (ADS)

The quantum instanton approximation is used to compute kinetic isotope effects for intramolecular hydrogen transfer in cis-1,3-pentadiene. Due to the importance of skeleton motions, this system with 13 atoms is a simple prototype for hydrogen transfer in enzymatic reactions. The calculation is carried out using thermodynamic integration with respect to the mass of the isotopes and a path integral Monte Carlo evaluation of relevant thermodynamic quantities. Efficient ``virial'' estimators are derived for the logarithmic derivatives of the partition function and the delta-delta correlation functions. These estimators require significantly fewer Monte Carlo samples since their statistical error does not increase with the number of discrete time slices in the path integral. The calculation treats all 39 degrees of freedom quantum mechanically and uses an empirical valence bond potential based on a molecular mechanics force field.

Vaní?ek, Ji?í; Miller, William H.



Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway.  


Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-(13)C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field (13)C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C. Brocadia fulgida synthesizes C(16:0) and isoC(16:0) fatty acids according to the known pathway of type II fatty acid biosynthesis. The (13)C-labelling pattern of the C(8) alkyl chain of the C(20) [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis. PMID:19175409

Rattray, Jayne E; Geenevasen, Jan A J; van Niftrik, Laura; Rijpstra, W Irene C; Hopmans, Ellen C; Strous, Marc; Schouten, Stefan; Jetten, Mike S M; Sinninghe Damsté, Jaap S



Principles and Results of Stable Isotope Labelling of L-?-Aminoacids by Combined Chemical and Enzymatic Methods  

Microsoft Academic Search

The major routes for making aminoacids and their isotopomers described in the literature are briefly compiled, especially considering about fourty research papers from the last five years. This report concentrates on the introduction of H, C, N, O and O into different defined positions of L-?-aminoacids. A scheme of four reaction categories with flowcharts is presented (homologation reactions with labelled

F. J. Winkler; K. Kühnl; R. Medina; R. Schwarz Kaske; H. L. Schmidt



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

Microsoft Academic Search

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

Farhad Daghighian; John C. Mazziotta; Edward J. Hoffman; Peter Shenderov; Behzad Eshaghian; Stefan Siegel; Michael E. Phelps



Use of Differential Isotopic Labeling and Mass Spectrometry To Analyze Capacitation-Associated Changes in the Phosphorylation Status of Mouse Sperm Proteins  

PubMed Central

Mammalian sperm need to reside in the female reproductive tract for a finite period of time before acquiring fertilizing competence. The biochemical changes associated with this process are collectively known as “capacitation”. With the use of the mouse as an experimental model, we have previously demonstrated that capacitation is associated with a cAMP-dependent increase in protein tyrosine phosphorylation. However, little is known about the identity and function of the protein targets of this phosphorylation cascade. In the present work, we have used differential isotopic labeling coupled with immobilized metal affinity chromatography (IMAC)-based phosphopeptide enrichment and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer to measure the changes in protein phosphorylation resulting from the capacitation process. As no kinase activators and/or phosphatase inhibitors were used in the preparation of the sperm samples, phosphorylated residues identified in this study represent in vivo sites of phosphorylation. Also, in contrast to other methods which rely on the incorporation of isotopically labeled amino acids at the protein level (e.g., SILAC), the present technique is based on the Fisher esterification of protein digests, allowing for the comparison of phosphorylation status in the absence of protein synthesis. This approach resulted in the identification of 55 unique, in vivo sites of phosphorylation and permitted the relative extent of phosphorylation, as a consequence of capacitation, to be calculated for 42 different phosphopeptides. This work represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process.

Platt, Mark D.; Salicioni, Ana M.; Hunt, Donald F.; Visconti, Pablo E.



Potential bias and mitigations when using stable isotope labeled parent drug as internal standard for LC-MS/MS quantitation of metabolites.  


In recent years, increasing emphasis has been placed on quantitative characterization of drug metabolites for better insight into the correlation between metabolite exposure and toxicological observations or pharmacological efficacy. One common strategy for metabolite quantitation is to adopt the stable isotope labeled (STIL) parent drug as the internal standard in an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. In the current work, we demonstrate this strategy could have a potential pitfall resulting in quantitation bias if the internal standard is subject to ion suppression from the co-eluting parent drug in the incurred samples. Propranolol and its metabolite 4-hydroxypropranolol were used as model compounds to demonstrate this phenomenon and to systematically evaluate different approaches to mitigate the issue, including atmospheric pressure chemical ionization (APCI) mode of ionization, increased internal standard concentration, quantitation without internal standard, the use of a structural analog as internal standard, and dilution of the samples. Case studies of metabolite quantitation in nonclinical and clinical studies in drug development were also included to demonstrate the importance of using an appropriate bioanalytical strategy for metabolite quantitation in the real world. We present that bias of metabolite concentrations could pose a potential for poor estimation of safety risk. A strategy for quantitation of metabolites in support of drug safety assessment is proposed. PMID:21056016

Jian, Wenying; Edom, Richard W; Xu, Yaodong; Gallagher, Joseph; Weng, Naidong



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.



Determination of isotopic ratios of L-leucine and L-phenylalanine and their stable isotope labeled analogues in biological samples by gas chromatography/triple-stage quadrupole mass spectrometry.  


A gas chromatographic/triple-stage quadrupole mass spectrometric (GC/MS/MS) method for measuring very low levels of enrichment of [5,5,5-2H3]-L-leucine and [ring-13C6]-L-phenylalanine in plasma and lipoprotein hydrolysates is described. The amino acids were derivatized to their N-heptafluorobutyryl isobutyl ester derivatives and the isotope ratio was determined by GC/MS/MS in the negative-ion chemical ionization mode. Parent ions were the [M-HF]- ions and fragment ions used for quantification were [P-2HF-C3H7]- (leucine) and [P-HF-OC4H9]- (phenylalanine), respectively. The limit of quantification was about 10 pg of the labeled compound co-eluting with 20 ng of the endogenous compound. The calibration curves were linear in the investigated range from 0.1% to 100% of the labeled compound. In biological samples, the higher selectivity of GC/MS/MS compared with GC/MS was demonstrated. PMID:8799305

Schweer, H; Watzer, B; Seyberth, H W; Steinmetz, A; Schaefer, J R



Probing the electronic structure of tyrosine radical Y Drad in photosystem II by EPR spectroscopy using site specific isotope labelling in Spirodela oligorrhiza  

NASA Astrophysics Data System (ADS)

Tyrosine (Y D) in the D2 reaction centre polypeptide of photosystem II (PSII) is redox-active and, under illumination, forms a dark-stable radical Y Drad . The origin of its stability and the functional role of Y Drad are not well understood. For understanding the electronic structure and reactivity of Y Drad , it is crucial to unambiguosly establish its hyperfine structure. There is considerable variation in the hyperfine data of Y Drad and their interpretation in literature. In the present study, the hyperfine structure of tyrosine radical Y Drad in PSII was probed by EPR in conjunction with carefully designed site specific isotope labelling. A comprehensive series of different selectively 2H-, 13C- or 17O-labeled tyrosine were synthesized and incorporated in Spirodela oligorrhiza with more than 95% enrichment. The 13C- and 17O-hyperfine interactions were obtained from spectral simulations. From the anisotropy of the hyperfine interactions the spin densities at all phenoxyl ring positions were precisely obtained. Comparison of the absolute differences in individual spin densities between Y Drad and neutral tyrosine radical in vitro with those of computationally calculated spin densities yield excellent agreement for a well ordered hydrogen bond between Y Drad and the surrounding protein matrix with a bond length of 1.5 Å. Enantioselective labeling confirms that the ?-methylene hydrogens of Y Drad in S. oligorrhiza are oriented in a highly constrained specific position making Y Drad strongly immobilized, thereby ensuring a firm hydrogen bond of the phenoxyl oxygen to the protein matrix.

Alia; Hulsebosch, Bob; van Gorkom, Hans J.; Raap, Jan; Lugtenburg, Johan; Matysik, Jörg; de Groot, Huub J. M.; Gast, Peter



Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris.  


Cost effective 13C/15N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The 13C/15N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut+) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (MutS) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for 13C-isotope labeling can be substantially reduced using the Mut+ strain compared to the MutS strain, as no 13C3-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for 15N-labeling by an optimal supply of 10 g/L (15NH4)2SO4 every 24 h. PMID:11519748

van den Burg, H A; de Wit, P J; Vervoort, J



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.

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



Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards and Labeling Programs for Clothes Washers, Water Dispensers, Vending Machines and CFLs  

Microsoft Academic Search

Since the late 1970s, energy labeling programs and mandatory energy performance standards have been used in many different countries to improve the efficiency levels of major residential and commercial equipment. As more countries and regions launch programs covering a greater range of products that are traded worldwide, greater attention has been given to harmonizing the specific efficiency criteria in these

David Fridley; Nina Zheng; Nan Zhou



Osmium isotopes suggest fast and efficient mixing in the oceanic upper mantle.  

NASA Astrophysics Data System (ADS)

The depleted upper mantle (DUM; the source of MORB) is thought to represent the complementary reservoir of continental crust extraction. Previous studies have calculated the "average" DUM composition based on the geochemistry of MORB. However the Nd isotope compositions of abyssal peridotites have been shown to extend to more depleted compositions than associated MORB. While this argues for the presence of both relatively depleted and enriched material within the upper mantle, the extent of compositional variability, length scales of heterogeneity and timescales of mixing in the upper mantle are not well constrained. Model calculations show that 2Ga is a reasonable mean age of depletion for DUM while Hf - Nd isotopes show the persistence of a depleted terrestrial reservoir by the early Archean (3.5-3.8Ga). U/Pb zircon ages of crustal rocks show three distinct peaks at 1.2, 1.9, and 2.7Ga and these are thought to represent the ages of three major crustal growth events. A fundamental question therefore is whether the present day upper mantle retains a memory of multiple ancient depletion events, or has been effectively homogenized. This has important implications for the nature of convection and time scales of survival of heterogeneities in the upper mantle. Here we compare published Os isotope data from abyssal peridotites and ophiolitic Os-Ir alloys with new data from Hawaiian spinel peridotite xenoliths. The Re-Os isotope system has been shown to yield useful depletion age information in peridotites, so we use it here to investigate the distribution of Re-depletion ages (TRD) in these mantle samples as a proxy for the variability of DUM. The probability density functions (PDF) of TRD from osmiridiums, abyssal and Hawaiian peridotites are all remarkably similar and show a distinct peak at 1.2-1.3 Ga (errors for TRD are set at 0.2Ga to suppress statistically spurious age peaks). The Hawaiian peridotites further show a distinct peak at 1.9-2Ga, but no oceanic mantle samples with TRD older than 2Ga have been reported. The TRD age peaks overlap with two major crustal building events recorded in the U/Pb crustal zircon ages. Therefore, peridotites from the convecting upper mantle can retain some memory of ancient depletion events, and these depletions are perhaps linked to major crustal building or large-scale mantle melting events. In the case of the Hawaiian peridotites, an ancient depletion event is further supported by some extremely radiogenic Hf isotope compositions. However, the vast majority of oceanic mantle samples show a narrow rage of Os isotope compositions (187Os/188Os = 0.123-0.126) with TRDs at 300-600 Ma. If the upper mantle has been produced continuously (or episodically) since at least the early Archean, it is then surprising that almost all oceanic mantle samples record such young depletion ages. We suggest that convective mixing in the mantle is rigorous enough that effectively re-homogenizes and resets the Os isotope composition of previously depleted peridotites within short time scales (<500Ma). Similarly recent ages have been derived from modeling the Sr, Nd, Hf, Pb isotopic composition of MORBs. This resetting and homogenization can be due to re-equilibration of depleted mantle with enriched components, e.g. recycled basaltic crust or more fertile mantle. Ancient depletion events are only effectively preserved in the sublithospheric mantle samples (e.g. Kaapval, Slave, Wyoming cratons) because they remain isolated from the convective mantle.

Bizimis, Michael; Salters, Vincent



Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo  

PubMed Central

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.

Tanrikulu, I. Caglar; Schmitt, Emmanuelle; Mechulam, Yves; Goddard, William A.; Tirrell, David A.



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.

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



An Efficient Method for Site-specific 18F-Labeling of Biomolecules Using the Rapid Condensation Reaction between 2-Cyanobenzothiazole and Cysteine  

PubMed Central

An efficient method based on a rapid condensation reaction between 2–cyanobenzothiazole (CBT) and cysteine has been developed for 18F–labeling of N–terminal cysteine–bearing peptides and proteins. An 18F–labeled dimeric cRGD ([18F]CBTRGD2) has been synthesized with an excellent radiochemical yield (92% based on radio–HPLC conversion, 80% decay–corrected and isolated yield) and radiochemical purity (>99%) under mild conditions using 18F–CBT, and shown good in vivo tumor targeting efficiency for PET imaging. The labeling strategy was also applied to the site–specific 18F–labeling of a protein, Renilla lucifierase (RLuc8) with a cysteine residue at its N–terminus. The protein labeling was achieved with 12% of decay–corrected radiochemical yield and more than 99% radiochemical purity. This strategy should provide a general approach for an efficient and site–specific 18F–labeling of various peptides and proteins for in vivo molecular imaging applications.

Jeon, Jongho; Shen, Bin; Xiong, Liqin; Miao, Zheng; Lee, Kyung Hyun; Rao, Jianghong; Chin, Frederick T.



Comprehensive and highly sensitive urinary steroid hormone profiling method based on stable isotope-labeling liquid chromatography-mass spectrometry.  


Steroid hormones are crucial substances that mediate a wide range of vital physiological functions of the body. Determination of the levels of steroid hormones plays an important role in understanding the mechanism of the steroid hormone-related diseases. In this study, we present a novel targeted metabolic profiling method based on the introduction of an easily protonated stable isotope tag to a hydroxyl-containing steroid hormone with a synthesized derivatization reagent, deuterium 4-(dimethylamino)-benzoic acid (d(4)-DMBA), and liquid chromatography-mass spectrometry (LC-MS). Different from other reported derivatization reagents that have been used to enhance the sensitivities for estrogens or androgens, our method is comprehensive with the capability of covering hydroxyl-containing androgens, estrogens, corticoids, and progestogens. Furthermore, the nonderivatized steroid hormones (e.g., 17?-hydroxyprogesterone, progesterone, and androstenedione) were not destroyed during the derivatization process, and their levels could still be obtained in one LC-MS run. We were able to detect 24 steroid hormones at subng/mL levels (the lower limit of detection could reach 5 pg/mL for estrone and 16?-hydroxy estrone, which is equivalent to 0.1 pg on column) with maximum sensitivity enhancement factors of more than 10(3)- to 10(4)-fold after derivatization. The method was successfully applied to the measurement of free (unconjugated) steroid hormones in urine samples of males, females, and pregnant women. Because the significant role the steroid hormone pathway plays in humans, a comprehensive, sensitive, specific, and accurate method for profiling the steroid hormone metabolome shall offer new insights into hormone-related diseases. PMID:23110480

Dai, Weidong; Huang, Qiang; Yin, Peiyuan; Li, Jia; Zhou, Jia; Kong, Hongwei; Zhao, Chunxia; Lu, Xin; Xu, Guowang



Noninvasive imaging of intracellular lipid metabolism in macrophages by Raman microscopy in combination with stable isotopic labeling.  


Monocyte-derived macrophages play a key role in atherogenesis because their transformation into foam cells is responsible for deposition of lipids in plaques within arterial walls. The appearance of cytosolic lipid droplets is a hallmark of macrophage foam cell formation, and the molecular basics involved in this process are not well understood. Of particular interest is the intracellular fate of different individual lipid species, such as fatty acids or cholesterol. Here, we utilize Raman microscopy to image the metabolism of such lipids and to trace their subsequent storage patterns. The combination of microscopic information with Raman spectroscopy provides a powerful molecular imaging method, which allows visualization at the diffraction limit of the employed laser light and biochemical characterization through associated spectral information. In order to distinguish the molecules of interest from other naturally occurring lipids spectroscopically, deuterium labels were introduced. Intracellular distribution and metabolic changes were observed for serum albumin-complexed palmitic and oleic acid and cholesterol and quantitatively evaluated by monitoring the increase in CD scattering intensities at 0.5, 1, 3, 6, 24, 30, and 36 h. This approach may also allow for investigating the cellular trafficking of other molecules, such as nutrients, metabolites, and drugs. PMID:22954250

Matthäus, Christian; Krafft, Christoph; Dietzek, Benjamin; Brehm, Bernhard R; Lorkowski, Stefan; Popp, Jürgen



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))



Amide or Amine: Determining the Origin of the 3300 cm?1 NH Mode in Protein SFG Spectra Using 15N Isotope Labels  

PubMed Central

Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases a strong NH mode near 3300 cm?1 is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode we studied 15N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an ?-helical secondary structure (LK?14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. 15N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm?1 on SiO2 and 13 cm?1 on CaF2. This clearly shows the 3300 cm?1 NH feature is associated with side chain NH stretches and not with backbone amide modes.

Weidner, Tobias; Breen, Nicholas F.; Drobny, Gary P.; Castner, David G.



Biosynthesis of the Pyrimidine Moiety of Thiamine Independent of the PurF Enzyme (Phosphoribosylpyrophosphate Amidotransferase) in Salmonella typhimurium: Incorporation of Stable Isotope-Labeled Glycine and Formate  

PubMed Central

Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-13C]glycine and [13C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a purF mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.

Enos-Berlage, Jodi L.; Downs, Diana M.



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



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.

Long, Fei; Cho, Wonhwa; Ishii, Yoshitaka



Ash, Carbon Isotope Discrimination, and Silicon as Estimators of Transpiration Efficiency in Crested Wheatgrass  

Microsoft Academic Search

Breeding and selection for higher transpiration efficiency (W) has been hampered by tedious and costly methodology. Rapid and less costly methods are needed for screening W in plant improvement programmes. We report the relationship of ash, silicon (Si) concentration, and Si uptake to W in crested wheatgrass (Agropyron desertorum (Fischer ex Link) Schultes), an important C3 range grass in western

H. F. Mayland; D. A. Johnson; K. H. Asay; A USDA-ARS; B USDA-ARS


Infrared, Vibrational Circular Dichroism, and Raman Spectral Simulations for ?-Sheet Structures with Various Isotopic Labels, Interstrand, and Stacking Arrangements Using Density Functional Theory.  


Infrared (IR), Raman, and vibrational circular dichroism (VCD) spectral variations for different ?-sheet structures were studied using simulations based on density functional theory (DFT) force field and intensity computations. The DFT vibrational parameters were obtained for ?-sheet fragments containing nine-amides and constrained to a variety of conformations and strand arrangements. These were subsequently transferred onto corresponding larger ?-sheet models, normally consisting of five strands with ten amides each, for spectral simulations. Further extension to fibril models composed of multiple stacked ?-sheets was achieved by combining the transfer of DFT parameters for each sheet with dipole coupling methods for interactions between sheets. IR spectra of the amide I show different splitting patterns for parallel and antiparallel ?-sheets, and their VCD, in the absence of intersheet stacking, have distinct sign variations. Isotopic labeling by (13)C of selected residues yields spectral shifts and intensity changes uniquely sensitive to relative alignment of strands (registry) for antiparallel sheets. Stacking of multiple planar sheets maintains the qualitative spectral character of the single sheet but evidences some reduction in the exciton splitting of the amide I mode. Rotating sheets with respect to each other leads to a significant VCD enhancement, whose sign pattern and intensity is dependent on the handedness and degree of rotation. For twisted ?-sheets, a significant VCD enhancement is computed even for sheets stacked with either the same or opposite alignments and the inter-sheet rotation, depending on the sense, can either further increase or weaken the enhanced VCD intensity. In twisted, stacked structures (without rotation), similar VCD amide I patterns (positive couplets) are predicted for both parallel and antiparallel sheets, but different IR intensity distributions still enable their differentiation. Our simulation results prove useful for interpreting experimental vibrational spectra in terms of ?-sheet and fibril structure, as illustrated in the accompanying paper. PMID:23924300

Welch, William R W; Kubelka, Jan; Keiderling, Timothy A



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


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



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



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

SciTech Connect

A ThermoFisher 'Triton' multi-collector thermal ionization mass spectrometer (MC-TIMS) was evaluated for trace and ultra-trace level isotoperatioanalysis 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 (10{sup 4} atoms to 10{sup 5} atoms) for {sup 239-242+244}Pu, {sup 233+236}U, {sup 241-243}Am, {sup 89,90}Sr, and {sup 134,135,137}Cs, and {le} 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 x 10{sup 6} or better using a SEM are reported here. Precisions of RSD {approx} 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, Stefan [New Brunswick Laboratory, Argonne, IL; Riciputi, Lee R [Los Alamos National Laboratory (LANL); Bostick, Debra A [ORNL; Turgeon, Steven [University of Alberta, Edmondton, Canada; McBay, Eddie H [ORNL; Lavelle, Mark [ORNL



Y(BD4)3, an efficient store of deuterium, and impact of isotope effects on its thermal decomposition  

NASA Astrophysics Data System (ADS)

Y(BD4)3, which stores as much as 16.6 wt.% and 252 kg/m3 D, has been synthesized via high-energy disk milling. The thermal decomposition of Y(BD4)3 has been investigated using thermogravimetric and calorimetric analyses combined with the spectroscopic evolved gas analysis. Two major endothermic events corresponding to thermal decomposition could be distinguished in the DSC profile up to 400 °C at ca. 231 and 285 °C, preceded by a phase transition (at ca. 198 °C) from the low-temperature Pa-3 form to a high-temperature polymorph of Y(BD4)3 (F-43c). The high-temperature phase forming at the onset of thermal decomposition may be prepared quantitatively by heating of the low-temperature phase to ca. 216 °C followed by rapid quenching.Effects of isotope H?D substitution on various properties of yttrium borohydride have been analyzed. Y(BD4)3 constitutes a very efficient low-temperature source of deuterium gas on the laboratory scale.

Jaro?, Tomasz; Grochala, Wojciech



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



Quantitative phosphoproteomics studies using stable isotope dimethyl labeling coupled with IMAC-HILIC-nanoLC-MS/MS for estrogen-induced transcriptional regulation.  


17?-Estradiol (E2) regulates transcriptional activity partly by inducing protein-kinase cascades, leading to the phosphorylation of estrogen receptors (ERs) and other functional proteins. Many of these phosphorylation events are also modulated by growth factors. To gain an insight into E2-modulated protein phosphorylation, we applied quantitative phosphoproteomics to investigate global changes in protein phosphorylation induced by E2 in MCF-7 cells. Proteomic analyses using stable isotope dimethyl labeling coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography (IMAC-HILIC) fractionation and nanoLC-MS/MS identified and quantified 2857 unique phosphorylation sites in 1338 phosphoproteins from 1 mg of total cellular protein. In addition to S118 of ER?, a 30-min E2 treatment significantly altered the status of 403 phosphorylation sites, including 112 novel sites. Interestingly, the substrate motifs for ERK1/2 were largely enriched in both the up-regulated and down-regulated phosphorylation sites. An increase in the phosphorylation on either the T202 or Y204 sites of ERK1 was observed after E2 treatment, while dual phosphorylation on both sites were not detected, implying that a feedback loop to deactivate MAPK signaling was achieved during a 30-min E2 treatment. In contrast, the PKA and CKII substrate motifs were majorly enriched among the up-regulated phosphorylation sites. Western blot analysis confirmed that E2 increased the phosphorylation level of S226 within a CKII motif of HSP90? by a factor of 2- to 3-fold without changing the total protein expression level. E2 also up-regulated phosphorylations of S255 in HSP90? and S353 within a CKII motif of HSP90?. These results indicated that E2 may modulate gene transcription by affecting the stability, function, and activity of many regulators through a HSP90 phosphorylation-mediated chaperoning process. This study, using a quantitative, multidimensional separation phosphoproteomic approach that required a relatively low amount of cells, provides new insights into the diversity, variability, and dynamic nature of the protein phosphorylation/dephosphorylation elicited by E2. PMID:21210654

Wu, Chin-Jen; Chen, Yen-Wen; Tai, Jung-Hsiang; Chen, Shu-Hui



Shock tube measurements of the tert-butanol + OH reaction rate and the tert-C4H8OH radical ?-scission branching ratio using isotopic labeling.  


The overall rate constant for the reaction tert-butanol + OH ? products was determined experimentally behind reflected shock waves by using (18)O-substituted tert-butanol (tert-butan(18)ol) and tert-butyl hydroperoxide (TBHP) as a fast source of (16)OH. The data were acquired from 900 to 1200 K near 1.1 atm and are best fit by the Arrhenius expression 1.24 × 10(-10)?exp(-2501/T [K]) cm(3) molecule(-1) s(-1). The products of the title reaction include the tert-C4H8OH radical that is known to have two major ?-scission decomposition channels, one of which produces OH radicals. Experiments with the isotopically labeled tert-butan(18)ol also lead to an experimental determination of the branching ratio for the ?-scission pathways of the tert-C4H8OH radical by comparing the measured pseudo-first-order decay rate of (16)OH in the presence of excess tert-butan(16)ol with the respective decay rate of (16)OH in the presence of excess tert-butan(18)ol. The two decay rates of (16)OH as a result of reactions with the two forms of tert-butanol differ by approximately a factor of 5 due to the absence of (16)OH-producing pathways in experiments with tert-butan(18)ol. This indicates that 80% of the (16)OH molecules that react with tert-butan(16)ol will reproduce another (16)OH molecule through ?-scission of the resulting tert-C4H8(16)OH radical. (16)OH mole fraction time histories were measured using narrow-line-width laser absorption near 307 nm. Measurements were performed at the line center of the R22(5.5) transition in the A-X(0,0) band of (16)OH, a transition that does not overlap with any absorption features of (18)OH, hence yielding a measurement of (16)OH mole fraction that is insensitive to any production of (18)OH. PMID:23683356

Stranic, Ivo; Pang, Genny A; Hanson, Ronald K; Golden, David M; Bowman, Craig T



Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells*  

PubMed Central

The thyroid hormone, 3, 3?,5-triiodo-l-thyronine (T3), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T3 are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T3-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TR?1 (HepG2-TR?1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T3 target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions –327/–312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T3 induced PAI-1 expression in J7-TR?1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T3/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T3-treated HepG2-TR?1 cells. The T3-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T3-associated tumor progression and prognosis.

Chen, Cheng-Yi; Chi, Lang-Ming; Chi, Hsiang-Cheng; Tsai, Ming-Ming; Tsai, Chung-Ying; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Wei-Jan; Huang, Ya-Hui; Lin, Kwang-Huei



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



Efficiency of labelling of red blood cells with technetium-99m after dipyridamole infusion for thallium-201 stress testing  

Microsoft Academic Search

Experimental studies have suggested that dipyridamole may inhibit red blood cell labelling with technetium-99m. To evaluate whether this effect is clinically relevant to the performance of radionuclide ventriculography after dipyridamole-thallium stress testing, in vitro red blood cell labelling was compared immediately before and after thallium-201 stress scintigraphy combined with either dipyridamole infusion (30 patients) or exercise stress (20 patients). Modified

Rodney J. Hicks; Peter Eu; L. Barry Arkles



Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes  

Microsoft Academic Search

To investigate the feasibility of conducting a genomic-scale protein labeling and localization study in Escherichia coli, a representative subset of 23 coding DNA sequences (CDSs) was selected for chromosomal tagging with one or more fluorescent protein genes (EGFP, EYFP, mRFP1, DsRed2). We used -Red recombination to precisely and efficiently position PCR-generated DNA target- ing cassettes containing a fluorescent protein gene

Rory M. Watt; Jing Wang; Meikid Leong; Hsiang-fu Kung; Kathryn S. E. Cheah; Depei Liu; Antoine Danchin; Jian-Dong Huang



Water use Efficiency in a Blue oak ( Quercus douglasii) Savanna - a Combined Analysis of Stable Isotopes and Eddy Covariance Measurements  

NASA Astrophysics Data System (ADS)

Understanding the relationship between carbon assimilation and water consumption by natural vegetation is needed to assess how changes in climate will affect plant carbon and water exchange as well as the energy fluxes of ecosystems. While climate change is expected to cause significant warming, most models also suggest changes in the timing and amount of precipitation received; thus implications of this type of change are particularly acute in Mediterranean regions of the world. Blue oak savannas are already exposed to broad variation in water availability and to severe droughts during the summer months. Our objective was to evaluate the trade-off between carbon gain and water loss (Water Use Efficiency) in this ecosystem at both the leaf and at the ecosystem scales. We monitored the ratio of the partial pressures of CO2 inside the leaf (Ci) and in the outside air (Ca) or Ci/Ca, during the summer months of three subsequent years. This ratio is determined by the balance between photosynthetic capacity and stomatal conductance to water loss. Leaf-level estimates for individual trees were based on the carbon isotope composition (?13C) of bulk leaf tissue and of recently fixed carbohydrates (leaf soluble sugars). These leaf and individual tree based estimates were then compared with canopy-level estimates derived from continuous eddy covariance measurements of fluxes of CO2, water vapor and meteorological variables from two eddy covariance systems, one above (23m) and one below (2m) the tree canopy. We found that savanna Blue oak trees cope with severe drought through coordinated down-regulation of carbon and water fluxes, i.e. the ratio Ci/Ca remained stable over four summer months, despite decreasing soil water content and leaf water potentials. Stable C isotope composition of leaf soluble sugars is the most robust measure of Ci/Ca because it reflects the initial discrimination of photosynthetic products, without the confounding effects ascribed to storage, tissue chemical composition and time of tissue formation. Our findings at the leaf-level were confirmed at the ecosystem-level by using a two tower (above and below canopy) eddy covariance method.

Mambelli, S.; Tu, K. P.; Knohl, A.; Ma, S.; Baldocchi, D. D.; Dawson, T. E.



Estimating Water Use Efficiency at the Watershed Scale Using Stable Isotopes  

NASA Astrophysics Data System (ADS)

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 ?13C throughout the cold air profile at the base of a constrained watershed could provide an estimate of ecosystem WUE. Because cold air profiles are very deep in complex terrain, they are difficult to sample. We used a tethered helium balloon and attached tubing to investigate the potential of using nocturnal cold air drainage to estimate ecosystem WUE at the watershed scale. The balloon was launched at the base of a constrained forested watershed in Northern Idaho. We monitored air temperature, CO2 concentration and ?13C from 0.1m to 206m on July 22, Aug 16 and Aug 27 , 2006. The inversion was deep, frequently reaching 166m, with observed lapse rates of 63.0, 65.0, and 54.0 °C/km. On the same sample dates, CO2 concentrations ranged from approx. 385 ppm at the top of the profile to 460 ppm at 1m. The ?13C typically ranged from -8.4 ‰ to -11.0 ‰ from 206 to 1m respectively. This range of CO2 concentrations (> 60 ppm) was sufficient for "Keeling plot" analysis and ecosystem respired ?13C was estimated as -24.49, -24.78 and -24.89 ‰. These values matched the mean soil respired CO2 ?13C of -25.0 ‰ (SD=0.98) measured at 40 points in the watershed on Aug 18. These measurements were made during a pronounced seasonal drought and when maximum vapor pressure deficit exceeded 2 kPa almost every day. After the drought breaks in the fall, we will determine if this sampling method is robust enough to detect shifts in ?13C due to soil water availability and declining vapor pressure deficits.

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



Cosmetic Labeling & Label Claims  


... to all labels and other written, printed, or graphic matter on or accompanying a product [FD&C ... drug and cosmetic ingredient labeling [21 CFR 701.3(d)]. The drug ingredients must appear according to the ...


Stable Carbon Isotope Composition (?13C), Water Use Efficiency, and Biomass Productivity of Lycopersicon esculentum, Lycopersicon pennellii, and the F1 Hybrid  

PubMed Central

Three tomatoes, Lycopersicon esculentum Mill. cv UC82B, a droughttolerant wild related species, Lycopersicon pennellii (Cor.) D'Arcy, and their F1 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 H2O) 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 F1 hybrid. Instantaneous water-use efficiency (micromole CO2/millimole H2O) 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.

Martin, Bjorn; Thorstenson, Yvonne R.



Estimation of the efficiency of hydrocarbon mineralization in soil by measuring CO2-emission and variations in the isotope composition of carbon dioxide  

Microsoft Academic Search

Estimation of the efficiency of hydrocarbon mineralization in soil by measuring CO2-emission and variations in the isotope composition of carbon dioxide E. Dubrovskaya1, O. Turkovskaya1, A. Tiunov2, N. Pozdnyakova1, A. Muratova1 1 - Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, Saratov, 2 - A.N. Severtsov Institute of Ecology and Evolution, RAS, Moscow, Russian Federation Hydrocarbon mineralization in

Ekaterina Dubrovskaya; Olga Turkovskaya



An Efficient and Cost-Effective Protocol for Selecting Transcription Factor Binding Sites that Reduces Isotope Usage  

PubMed Central

To function, transcription factors must position themselves by binding to DNA in a sequence-specific manner. Knowing the binding sites of these factors is a necessary step in understanding their activity. The standard protocols used for selecting a consensus-binding sequence for a DNA binding domain often require the use of radioisotopes to attain the necessary level of power in the assay. Alternatives are often less sensitive and may require an expensive apparatus for visualizing. We have created a modified binding site selection (BSS) protocol to improve efficiency and decrease the use of radioisotope. A GST affinity-tagged DNA binding domain construct was immobilized on a GSH affinity column and used to select from a randomized oligonucleotide library identical to those typically used in a radiolabeled BSS protocol. This produced a library specifically pre-enriched for use in a standard sequential EMSA selection. Use of a pre-enriched library reduced the total number of labeled rounds required for selection, decreasing the use of radioisotope while maintaining efficacy. The protocol was used to select for the binding sequence for several Drosophila melanogaster transcription factors. The consensus sequence was then shown by competitive binding experiments to associate with the protein in a sequence-dependent manner.

Krystel, Joseph; Ayyanathan, Kasirajan



Europium-labeled activity-based probe through click chemistry: absolute serine protease quantification using (153)Eu isotope dilution ICP/MS.  


Click and analyze: the titled probe was synthesized by conjugating a sulfonyl fluoride and azido unit using click chemistry to give SF-Eu, which can react specifically with serine (Ser) in the active site of serine protease (SP). Combination of the method with (153)Eu-isotope dilution ICP/MS enables absolute protein quantification of active SPs in biological samples using only one (153)Eu(NO(3))(3) isotopic standard. PMID:22344943

Yan, Xiaowen; Luo, Yacui; Zhang, Zhubao; Li, Zhaoxin; Luo, Qiang; Yang, Limin; Zhang, Bo; Chen, Haifeng; Bai, Peiming; Wang, Qiuquan



Development of a stable isotope dilution assay for an accurate quantification of protein-bound N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine using a (13)C-labeled internal standard.  


Syntheses of the labeled Amadori compound [(13)C(6)]-N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine ([(13)C(6)]-DFLys) and the labeled glycated tetrapeptide Ala-[(13)C(6)]-DFLys-Leu-Gly are presented. The compounds were used in the development of stable isotope dilution assays for the quantification of the degree of glycosylation of bovine serum albumin treated for 20 min at 95 degrees C in the presence of glucose. The experiments revealed that the use of the labeled standards in combination with LC/MS allowed the exact quantification of protein-bound DFLys with the high recovery rate of 95% (at a spike level of 150 nmol/mg of protein) and a low detection limit of 5 nmol/mg of protein. The data revealed, however, that DFLys is significantly degraded during the enzymic hydrolysis of the protein backbone generally needed in the quantification procedure and, furthermore, incomplete digestion of the protein was observed. Both sources of errors were clearly overcome by using in particular the labeled peptide as the internal standard. PMID:10606577

Vinale, F; Fogliano, V; Schieberle, P; Hofmann, T



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



The isotopic composition of H2 from biomass burning - dependency on combustion efficiency, moisture content and dD of local precipitation  

NASA Astrophysics Data System (ADS)

Differences in isotopic composition between the various sources of H2 are large, but only few measurements have been carried out to constrain them. For biomass burning, the values quoted in the literature are based on few combustion experiments, which were then extrapolated to the global scale based on a number of assumptions. One of these assumptions is that the isotopic composition of H2 should scale with the isotopic composition of the precipitation at the location where the biomass grew. Here we test this hypothesis using 18 wood samples collected from various locations around the globe. The sample locations cover a range in dD of precipitation from below -120 permil in Siberia and Canada to -15 permil in Zimbabwe. The results confirm the predicted linear relation with dD of the precipitation in the sampling region. The water content itself is found to at most slightly affect the results. Furthermore, dD of H2 depends on combustion efficiency. Thus, the isotopic composition of H2 from biomass burning shows a strong variability around the globe, and between different stages of a fire. It is suggested that this variability, rather than a global bulk number, should be incorporated explicitly in global models that attempt to reproduce the spatial and temporal distribution of dD in H2.

Röckmann, Thomas; Gómez Álvarez, Catalina; Walter, Sylvia; Wollny, Adam; Gunthe, Sachin; Helas, Günter; Pöschl, Ulrich; Keppler, Frank; Greule, Markus; Brand, Willi



Does a stable isotopically labeled internal standard always correct analyte response? A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma.  


A stable isotopically labeled (SIL) analogue is believed to be the most appropriate internal standard in a quantitative bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay. It is assumed that a SIL internal standard always compensates for variability in chemical derivatization, sample extraction and LC/MS/MS analysis due to its nearly identical chemical and physical properties to the unlabeled analyte. Hence, the analyte to internal standard peak area ratio should be constant despite any variations in sample processing or analysis. However, in our laboratories, a deuterium labeled internal standard of carvedilol demonstrated an unexpected behavior-the analyte to internal standard peak area ratio changed with two specific lots of commercially supplied human plasma. Several experiments, including dilution of the extract with LC mobile phase and post-column infusion of the carvedilol solution followed by the injection of extracted blank plasma, have indicated that a high level of matrix suppression affected the ionization of the carvedilol-S enantiomer and its deuterated internal standard differently in these two lots of plasma. For the first time, it was clearly demonstrated that a slight difference in retention time between the analyte and the SIL internal standard, caused by deuterium isotope effect, has resulted in a different degree of ion suppression between these two analogues. This difference was significant enough to change the analyte to internal standard peak area ratio and affect the accuracy of the method. PMID:16959461

Wang, Sherry; Cyronak, Matthew; Yang, Eric



New Approach via Gene Knockout and Single-Step Chemical Reaction for the Synthesis of Isotopically Labeled Fusarin C as an Internal Standard for the Analysis of this Fusarium Mycotoxin in Food and Feed Samples.  


The gold standard for quantitation of contaminants with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is the use of isotopically labeled standards. Herein, we report a new strategy for the synthesis of isotopically labeled 21-d(3)-fusarin C via a genetically modified Fusarium strain, followed by a one-step derivatization reaction. Fusarin C is a Fusarium mycotoxin, which is mutagenic after metabolic activation. Its occurrence has been demonstrated recently in corn-based samples, but up to now, little is known about the contamination of other grain samples. To collect further data, the quantitation method was enhanced by application of the 21-d(3)-fusarin C and the use of a QTRAP 5500 mass spectrometer. This new method has a limit of detection (LOD) of 1 ?g/kg, a limit of quantitation (LOQ) of 4 ?g/kg, and a recovery rate of 99%. A total of 21 corn samples and 13 grain samples were analyzed, with resulting fusarin C levels varying from not detectable to 24.7 ?g/kg. PMID:22877497

Kleigrewe, Karin; Niehaus, Eva-Maria; Wiemann, Philipp; Tudzynski, Bettina; Humpf, Hans-Ulrich



Determination of extremely low (236)U/(238)U isotope ratios in environmental samples by sector-field inductively coupled plasma mass spectrometry using high-efficiency sample introduction.  


A method by inductively coupled plasma mass spectrometry (ICP-MS) was developed which allows the measurement of (236)U at concentration ranges down to 3 x 10(-14)g g(-1) and extremely low (236)U/(238)U isotope ratios in soil samples of 10(-7). By using the high-efficiency solution introduction system APEX in connection with a sector-field ICP-MS a sensitivity of more than 5,000 counts fg(-1) uranium was achieved. The use of an aerosol desolvating unit reduced the formation rate of uranium hydride ions UH(+)/U(+) down to a level of 10(-6). An abundance sensitivity of 3 x 10(-7) was observed for (236)U/(238)U isotope ratio measurements at mass resolution 4000. The detection limit for (236)U and the lowest detectable (236)U/(238)U isotope ratio were improved by more than two orders of magnitude compared with corresponding values by alpha spectrometry. Determination of uranium in soil samples collected in the vicinity of Chernobyl nuclear power plant (NPP) resulted in that the (236)U/(238)U isotope ratio is a much more sensitive and accurate marker for environmental contamination by spent uranium in comparison to the (235)U/(238)U isotope ratio. The ICP-MS technique allowed for the first time detection of irradiated uranium in soil samples even at distances more than 200 km to the north of Chernobyl NPP (Mogilev region). The concentration of (236)U in the upper 0-10 cm soil layers varied from 2 x 10(-9)g g(-1) within radioactive spots close to the Chernobyl NPP to 3 x 10(-13)g g(-1) on a sampling site located by >200 km from Chernobyl. PMID:16504353

Boulyga, Sergei F; Heumann, Klaus G



Luminescent dye-doped or rare-earth-doped monodisperse silica nanospheres as efficient labels in DNA microarrays  

Microsoft Academic Search

Luminescent nanoparticles are gaining more and more interest in bio-labeling and bio-imaging applications, like for example DNA microarray. This is a high-throughput technology used for detection and quantification of nucleic acid molecules and other ones of biological interest. The analysis is resulting by specific hybridization between probe sequences deposited in array and a target ss-DNA usually expressed by PCR and

F. Enrichi; R. Riccò; A. Meneghello; R. Pierobon; F. Marinello; P. Schiavuta



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


Efficient Estimators for Quantum Instanton Evaluation of theKinetic Isotope Effects: Application to the Intramolecular HydrogenTransfer in Pentadiene  

SciTech Connect

The quantum instanton approximation is used to compute kinetic isotope effects for intramolecular hydrogen transfer in cis-1,3-pentadiene. Due to the importance of skeleton motions, this system with 13 atoms is a simple prototype for hydrogen transfer in enzymatic reactions. The calculation is carried out using thermodynamic integration with respect to the mass of the isotopes and a path integral Monte Carlo evaluation of relevant thermodynamic quantities. Efficient 'virial' estimators are derived for the logarithmic derivatives of the partition function and the delta-delta correlation functions. These estimators require significantly fewer Monte Carlo samples since their statistical error does not increase with the number of discrete time slices in the path integral. The calculation treats all 39 degrees of freedom quantum-mechanically and uses an empirical valence bond potential based on a modified general AMBER force field.

Vanicek, Jiri; Miller, William H.



Evidence for the biosynthetic pathway from sinapic acid to syringyl lignin using labeled sinapic acid with stable isotope at both methoxy groups in Robinia pseudoacacia and Nerium indicum.  


A tracer experiment using synthesized labeled lignin precursors was designed to confirm the actual biosynthetic pathway for syringyl lignin. Tetradeuteroferulic acid-[8-D, 3-OCD(3)] and heptadeuterosinapic acid-[8-D, 3,5-OCD(3)] were synthesized and fed to shoots of robinia (Robinia pseudoacacia) and oleander (Nerium indicum) trees. The incorporation of each labeled precursor into lignin was traced by gas chromatography-mass spectrometry. The synthesized sinapic acid, in which both methoxy groups were labeled, was useful in monitoring the conversion of sinapic acid into syringyl lignin. When heptadeuterosinapic acid was fed, syringyl units containing seven deuterium labels were detected. The results of this study support the traditionally accepted pathway that sinapic acid is converted to sinapyl alcohol via sinapoyl-CoA in robinia and oleander. PMID:12009990

Yamauchi, Kazuchika; Yasuda, Seiichi; Fukushima, Kazuhiko



Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards  

Microsoft Academic Search

A novel method was developed for the quantitative analysis of the microbial metabolome using a mixture of fully uniformly (U) 13C-labeled metabolites as internal standard (IS) in the metabolite extraction procedure the subsequent liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS\\/MS) analysis. This mixture of fully U 13C-labeled metabolites was extracted from biomass of Saccharomyces cerevisiae cultivated in a fed-batch fermentation on

Liang Wu; Mlawule R. Mashego; Jan C. van Dam; Angela M. Proell; Jacobus L. Vinke; Cor Ras; Wouter A. van Winden; Walter M. van Gulik; Joseph J. Heijnen



Production of stable-isotope-labeled bovine heme and its use to measure heme-iron absorption in children1-4  

Microsoft Academic Search

Background: The use of stable isotopes has provided valuable insights into iron absorption in humans, but the data have been limited to nonheme iron. Objective: Our objectives were to produce heme iron enriched in 58 Fe and to use it to study the absorption of heme iron and the effect of iron and zinc intakes on heme-iron absorption in children.

Paz Etcheverry; Gordon E Carstens; Erin Brown; Keli M Hawthorne; Zhensheng Chen; Ian J Griffin


Method of separating isotopes of uranium employing UO2(Hfacac)2. l with an improved photon efficiency  

SciTech Connect

Methods for separating uranium isotopes are disclosed including irradiating certain uranyl ion-containing compounds with radiation of a wavelength, lambda, at which the compounds have a predetermined absorption cross section sigma lambda at a power> kw/cm2 times sigma lambda /4 X 10-18 cm2.

Hall, R.B.



Eukaryotic expression system for the incorporation of stable isotopes into proteins  

US Patent & Trademark Office Database

Methods for producing stable isotope-labeled recombinant protein are provided. The methods include isolating a stable isotope-labeled recombinant protein from a Trichoplusia ni larva expressing a recombinant protein, which Trichoplusia ni larva has ingested a food source comprising stable isotope-labeled algae, thereby resulting in incorporation of a stable isotope into the recombinant protein to produce the stable isotope-labeled recombinant protein.

Kobilka; Brian (Palo Alto, CA); Bokoch; Michael (Menlo Park, CA)



Energy Efficiency in the Future Internet: The Role of Optical Packet Switching and Optical-Label Switching  

Microsoft Academic Search

This paper reviews the energy efficiency of optical- packet-switching (OPS) systems in comparison with electronic packet switching and hybrid packet switching in the context of future networks. The paper will first discuss the energy efficiency metrics that should include considerations for life-cycle analy- sis, applications, and network-wide goodput. The state-of-the-art electronic packet switching router is currently energy-limited in scalability as

S. J. Ben Yoo



On the reaction mechanism for hydrocarbon formation from methanol over SAPO-34: 1. Isotopic labeling studies of the Co-reaction of ethene and methanol  

Microsoft Academic Search

¹³C-Methanol and ¹²C-ethene (fed as ethanol) have been co-reacted over SAPO-34 in a flow system at 400°C using argon as a carrier (diluent) gas. The feed contained an equal number of ¹³C and ¹²C atoms. The products were analyzed by GC-MS, allowing the determination of the isotopic composition of the reactor effluent. The ethanol was immediately converted to ethene, so

I. M. Dahl; S. Kolboe



Determination of depleted uranium in urine via isotope ratio measurements using large-bore direct injection high efficiency nebulizer-inductively coupled plasma mass spectrometry.  


Inductively coupled plasma mass spectrometry (ICP-MS), coupled with a large-bore direct injection high efficiency nebulizer (LB-DIHEN), was utilized to determine the concentration and isotopic ratio of uranium in 11 samples of synthetic urine spiked with varying concentrations and ratios of uranium isotopes. Total U concentrations and (235)U/(238)U isotopic ratios ranged from 0.1 to 10 microg/L and 0.0011 and 0.00725, respectively. The results are compared with data from other laboratories that used either alpha-spectrometry or quadrupole-based ICP-MS with a conventional nebulizer-spray chamber arrangement. Severe matrix effects due to the high total dissolved solid content of the samples resulted in a 60 to 80% loss of signal intensity, but were compensated for by using (233)U as an internal standard. Accurate results were obtained with LB-DIHEN-ICP-MS, allowing for the positive identification of depleted uranium based on the (235)U/(238)U ratio. Precision for the (235)U/(238)U ratio is typically better than 5% and 15% for ICP-MS and alpha-spectrometry, respectively, determined over the concentrations and ratios investigated in this study, with the LB-DIHEN-ICP-MS system providing the most accurate results. Short-term precision (6 min) for the individual (235)U and (238)U isotopes in synthetic urine is better than 2% (N = 7), compared to approximately 5% for conventional nebulizer-spray chamber arrangements and >10% for alpha-spectrometry. The significance of these measurements is discussed for uranium exposure assessment of Persian Gulf War veterans affected by depleted uranium ammunitions. PMID:15479520

Westphal, Craig S; McLean, John A; Hakspiel, Shelly J; Jackson, William E; McClain, David E; Montaser, Akbar



On the reaction mechanism for hydrocarbon formation from methanol over SAPO-34. 2. Isotopic labeling studies of the Co-reaction of propene and methanol  

SciTech Connect

[{sup 13}C]Methanol and [{sup 12}C]propene (fed as isopropanol, which is immediately converted to propene) have been co-reacted over SAPO-34 in a flow system at 400{degrees}C using argon as a carrier gas. The feed was equimolar in {sup 13}C and {sup 12}C atoms. The products were analyzed by gas chromatography-mass spectrometry, allowing determination of the isotopic composition. While the methanol was completely or almost completely converted to hydrocarbons, the larger part of the propene emerged unreacted. The products ethen and butenes were mostly formed from methanol and contained a large excess of {sup 13}C atoms. The propene effluent consisted mainly of all- {sup 12}C or all-{sup 13}C molecules and, only to a small extent, isotopically mixed molecules. The tendency for propene to emerge unreacted and all new hydrocarbons to be formed from methanol became more pronounced with progressing catalyst deactivation. The results show that the higher hydrocarbons are, over this catalyst, not formed by successive methylations of bulk gas-phase propene. A previously proposed {open_quotes}carbon pool{close_quotes} mechanism can explain the gross effects seen in the product and isotopic distribution, but it is pointed out that the nonreactivity of propene in SAPO-34 may be caused by slow diffusion of propene in the pores. 16 refs., 5 figs., 4 tabs.

Dahl, I.M.; Kolboe, S. [Univ. of Oslo (Norway)



Analysis of metabolic pathways via quantitative prediction of isotope labeling patterns: a retrobiosynthetic 13C NMR study on the monoterpene loganin  

Microsoft Academic Search

The monoterpene loganin serves as a precursor in the biosynthetic pathways of numerous indole alkaloids. In contrast to earlier studies, we present evidence that the biosynthesis of loganin in Rauwolfia serpentina cells proceeds mainly via the deoxyxylulose pathway and not by the mevalonate pathway. This conclusion is based on experiments using a R. serpentina cell culture supplied with 13C-labeled samples

Dietmar Eichinger; Adelbert Bacher; Meinhart H Zenk; Wolfgang Eisenreich



Efficiency in Programming for Generalization: Comparison of Two Methods for Teaching Expressive Labeling to Preschoolers with Developmental Delays  

Microsoft Academic Search

An adapted alternating treatments design was used to compare the efficiency of two methods of programming for generalization. Four preschool subjects with developmental delays were taught expressive identification of object pictures. During Condition I, single exemplars for each of two objects were trained to acquisition successively until generalization criterion was met. During Condition II, five exemplars for each of two

Carol M. Schroeder; John W. Schuster; Mary Louise Hemmeter



Cross-Strand Coupling and Site-Specific Unfolding Thermodynamics of a Trpzip ?-Hairpin Peptide Using 13 C Isotopic Labeling and IR Spectroscopy  

Microsoft Academic Search

Conformational properties of a 12-residue tryptophan zipper (trpzip) ? -hairpin peptide (AWAWENGKWAWK- NH2, a modification of the original trpzip2 sequence) are analyzed under equilibrium conditions using ECD and IR spectra of a series of variants, singly and doubly C1-labeled with 13C on the amide CdO. The characteristic features of the 13CdO component of the amide IIR band and their sensitivity

Rong Huang; Ling Wu; Dan McElheny; Petr Bour?; Anjan Roy; Timothy A. Keiderling



Characterization of n-butyl acrylate centered triads in poly(n-butyl acrylate-co-carbon monoxide-co-ethylene) by isotopic labeling and two dimensional NMR.  


Poly(n-butylacrylate-co-carbon monoxide-co-ethylene) (polyEBC) samples prepared from 13C-labeled monomer, n-butyl acrylate, were characterized using two dimensional (2D) pulsed field gradient (PFG) 750 MHz NMR spectroscopy. To elucidate the complex structure of the terpolymer, 2D-1H/13C-heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were conducted by selectively exciting the enhanced resonances in the spectra of two polymer samples, one polymer resulting from synthesis with 1-13C-n-butylacrylate monomer and a second polymer obtained from a synthesis with 2-13C-n-butylacrylate monomer. High-resolution 2D-NMR combined with 13C-labeling of the polymer greatly simplifies the 2D-NMR spectra, selectively enhances the weak peaks from low occurrence B-centered triad structures, and aids in their resonance assignments. In all experiments, the sample temperature was 120 degrees C, to ensure a homogeneous solution and sufficient molecular mobility. Electronic Supplementary Material: Supplementary material (1D 13C NMR spectra of the 13C-labeled and unlabeled polymers) is available in the online version of this article at PMID:15214405

Monwar, Masud; Oh, Sung Joon; Rinaldi, Peter L; McCord, Elizabeth F; Hutchinson, Robin A; Buback, Michael M; Latz, Henning



Efficient synthesis and chiral separation of 11C-labeled ibuprofen assisted by DMSO for imaging of in vivo behavior of the individual isomers by positron emission tomography.  


The pharmacological mechanisms focusing on chiral isomer of ibuprofen are not fully understood. Only the (S)-isomer of ibuprofen inhibits cyclooxygenases, which mediates the generation of prostanoids and thromboxanes. Consequently, (S)-isomers represent a major promoter of the anti-inflammatory effect, and the effects of the (R)-isomers have not been widely discussed. However, more recently, the cyclooxygenase-independent pharmacological effects of ibuprofen have been elucidated. Pharmacokinetic studies with individual isomers of ibuprofen by positron emission tomography should aid our understanding of the pharmacological mechanisms of ibuprofen. The efficient (11)C-labeling of ibuprofen for chiral separation via the TBAF-promoted ?-[(11)C]methylation was achieved by using DMSO rather than THF as the reaction solvent. The robust production of the radiochemically labile (11)C-labeled ibuprofen ester was realized by the protective effect of DMSO on radiolysis. After intravenous injection of each enantiomer of [(11)C]ibuprofen, significantly high radioactivity was observed in the joints of arthritis mice when compared to the levels observed in normal mice. However, the high accumulation was equivalent between the enantiomers, indicating that ibuprofen is accumulated in the arthritic joints regardless of the expression of cyclooxygenases. PMID:21515058

Kikuchi, Tatsuya; Okada, Maki; Nengaki, Nobuki; Furutsuka, Kenji; Wakizaka, Hidekatsu; Okamura, Toshimitsu; Zhang, Ming-Rong; Kato, Koichi



Investigation of Therapeutic Efficiency of Bleomycin and Bleomycin-Glucuronide Labeled with (131)I on the Cancer Cell Lines.  


Abstract The aim of this study is to determine the incorporations of radiolabeled bleomycin ((131)I-BLM) and bleomycin-glucuronide ((131)I-BLMGLU) on PC-3 (human prostate carcinoma cell line), Caco-2 (human colon adenocarcinoma cell line), Hutu-80 (Human Duodenum adenocarcinoma cell line), and A549 (Human lung adenocarcinoma epithelial cell line) cancerous cell lines. For this purpose, BLM and BLMGLU enyzmatically synthesized were labeled with (131)I, quality control studies were done and the incorporation yields of (131)I-BLM and (131)I-BLMGLU on these cell lines were measured. Quality-control studies showed that the radiolabeling yields were obtained as 95% and 90% for (131)I-BLM and (131)I-BLMGLU, respectively. Also, as a result of the cell culture studies, it was found that (131)I-BLM and (131)I-BLMGLU had higher incorporation on PC-3 cells than that of other cell lines. In addition to this, it was reported that the incorporation yield of (131)I-BLMGLU was higher than that (131)I-BLM. At the end of the study, cytotoxicities of BLM and BLMGLU on PC-3 cancerous cell line were inspected and fluorescent images of BLM and BLMGLU were taken on PC-3 cells by using fluorescein isothiocyanate. In conclusion, cell culture studies demonstrated that the incorporation values of (131)I-BLMGLU on the four cell lines were about five to six times higher than (131)I-BLM. Radiolabeled glucuronide derivatives can be used in cancer therapy and tumor imaging, depending on the properties of radioiodine for the ?-glucuronidase-rich tissues because glucuronidation leads to rapid and higher incorporation on adenocarcinoma cells. PMID:23350895

Ediz, Melis; Avc?ba??, U?ur; Unak, Perihan; Müftüler, Fazilet Zümrüt Biber; Medine, Emin ?lker; Yurt K?lçar, Ayfer; Demiro?lu, Hasan; Gümü?er, Fikriye Gül; Sakarya, Serhan



In Folio Respiratory Fluxomics Revealed by 13C Isotopic Labeling and H/D Isotope Effects Highlight the Noncyclic Nature of the Tricarboxylic Acid "Cycle" in Illuminated Leaves1[W  

PubMed Central

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

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



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.

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



On the reaction mechanism for hydrocarbon formation from methanol over SAPO-34: 1. Isotopic labeling studies of the Co-reaction of ethene and methanol  

SciTech Connect

{sup 13}C-Methanol and {sup 12}C-ethene (fed as ethanol) have been co-reacted over SAPO-34 in a flow system at 400{degrees}C using argon as a carrier (diluent) gas. The feed contained an equal number of {sup 13}C and {sup 12}C atoms. The products were analyzed by GC-MS, allowing the determination of the isotopic composition of the reactor effluent. The ethanol was immediately converted to ethene, so the reaction system was equivalent to a feed consisting of methanol/ethene/water. While the methanol was completely or almost completely converted to hydrocarbons, the larger part of the ethene emerged unreacted. The products propene and butenes were mostly formed from methanol and contained a large excess of {sup 13}C atoms. The ethene effluent consisted mainly of all {sup 12}C or all {sup 13}C atoms, and only to a small extent of {sup 12}C-{sup 13}C molecules. The reaction system was followed from an initially very active catalyst until the catalyst was sufficiently deactivated that C{sub 1} was not completely converted to hydrocarbons. The tendency for ethene to emerge unreacted, and for all new hydrocarbons to be formed from methanol became more pronounced with progressing catalyst deactivation. The results show clearly that the higher hydrocarbons are, over this catalyst, not formed by successive methylations of ethene. A previously proposed {open_quotes}carbon pool{close_quotes} mechanism can explain the gross effects seen in the product and isotopic distribution. 15 refs., 6 figs., 4 tabs.

Dahl, I.M.; Kolboe, S. [Univ. of Oslo (Norway)



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



/sup 18/O isotope shift in /sup 15/N NMR spectroscopy. 2. Synthesis of /sup 15/N, /sup 18/O-labeled hydroxylamine hydrochloride  

SciTech Connect

Since hydroxylamine can serve as a key intermediate in the synthesis of a variety of compounds, the synthesis of (/sup 15/N, /sup 18/O)-labelled hydroxylamine hydrochloride was undertaken. Published procedures for the synthesis of hydroxylamine resulted in poor yields in some cases and in lower percentage of /sup 18/O in the product than expected in other cases. The compound was synthesized in dry tetrahydrofuran (THF) by treating NaNO/sub 2/ with borane-methyl sulfide. The course of the reaction was examined using /sup 11/B NMR spectroscopy, and the product yield was 74%. The /sup 18/O enrichment was demonstrated by both mass spectrometry and /sup 15/N NMR of the isolated acetoxime. 23 references, 1 figure.

Rajendran, G.; Van Etten, R.L.



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



Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells  

Microsoft Academic Search

Quantitative immunoelectron microscopy often involves determining the distributions of gold label in different intracellular compartments and then drawing comparisons between compartments in the same sample of cells or between experimental groups of cells. In the case of within-group comparisons, recent developments in the estimation of relative labelling index and labelling density make it possible to test whether or not particular

Terry M. Mayhew; Gareth Griffiths; John M. Lucocq



The pharmacokinetic behavior of the soy isoflavone metabolite S-(-)equol and its diastereoisomer R-(+)equol in healthy adults determined by using stable-isotope-labeled tracers1234  

PubMed Central

Background: The nonsteroidal estrogen equol occurs as diastereoisomers, S-(?)equol and R-(+)equol, both of which have significant biological actions. S-(?)equol, the naturally occurring enantiomer produced by 20–30% of adults consuming soy foods, has selective affinity for estrogen receptor-?, whereas both enantiomers modulate androgen action. Little is known about the pharmacokinetics of the diastereoisomers, despite current interest in developing equol as a nutraceutical or pharmaceutical agent. Objective: The objective was to compare the pharmacokinetics of S-(?)equol and R-(+)equol by using [13C] stable-isotope-labeled tracers to facilitate the optimization of clinical studies aimed at evaluating the potential of these diastereoisomers in the prevention and treatment of estrogen- and androgen-dependent conditions. Design: A randomized, crossover, open-label study in 12 healthy adults (6 men and 6 women) compared the plasma and urinary pharmacokinetics of orally administered enantiomeric pure forms of S-(?)[2-13C]equol, R-(+)[2-13C]equol, and the racemic mixture. Plasma and urinary [13C]R-equol and [13C]S-equol concentrations were measured by tandem mass spectrometry. Results: Plasma [13C]equol concentration appearance and disappearance curves showed that both enantiomers were rapidly absorbed, attained high circulating concentrations, and had a similar terminal elimination half-life of 7–8 h. The systemic bioavailability and fractional absorption of R-(+)[2-13C]equol were higher than those of S-(?)[2-13C]equol or the racemate. The pharmacokinetics of racemic (±)[2-13C]equol were different from those of the individual enantiomers: slower absorption, lower peak plasma concentrations, and lower systemic bioavailability. Conclusions: The high bioavailability of both diastereoisomers contrasts with previous findings for the soy isoflavones daidzein and genistein, both of which have relatively poor bioavailability, and suggests that low doses of equol taken twice daily may be sufficient to achieve biological effects.

Zhao, Xueheng; Jha, Pinky; Heubi, James E; Brown, Nadine M



A General Cartographic Labeling Algorithm  

Microsoft Academic Search

Some apparently powerful algorithms for automatic label placement on maps use heuristicsthat capture considerable cartographic expertise but are hampered by provably inefficientmethods of search and optimization. On the other hand, no approach to label placement thatis based on an efficient optimization technique has been applied to the production of generalcartographic maps --- those with labeled point, line, and area features

Shawn Edmondson; Jon Christensen; Joe Marks; Stuart M. Shieber



Point labeling with sliding labels  

Microsoft Academic Search

This paper discusses algorithms for labeling sets of points in the plane, where labelsare not restricted to some nite number of positions. We show that continuously slidinglabels allow more points to be labeled both in theory and in practice. We denesix dierent models of labeling. We compare models by analyzing how many morepoints can receive labels under one model than

Marc J. Van Kreveld; Tycho Strijk; Alexander Wolff



Synthesis and NMR of RNA with selective isotopic enrichment in the bases.  

PubMed Central

Efficient syntheses of pyrimidine and purine nucleosides and nucleotides with selective 13C enrichment in the base moieties are described. Uridine and cytidine are labeled at position C6 and adenosine and guanosine are labeled at position C8. The selectively labeled nucleosides were converted to nucleoside triphosphates and used with in vitro transcription to synthesize labeled RNA. Isotope-edited 12C and 13C sub-spectra of a omega 1-1/2-X-filtered NOESY experiment are demonstrated to be useful for making resonance assignments and for deriving structural information in large (> 20 nt) RNA molecules. The labeled RNAs also allow heteronuclear J-couplings and relaxation parameters to be measured without complications from 13C-13C J-couplings.

SantaLucia, J; Shen, L X; Cai, Z; Lewis, H; Tinoco, I



Nitrifier denitrification can be a source of N2O from soil: a revised approach to the dual isotope labelling method  

NASA Astrophysics Data System (ADS)

Nitrous oxide (N2O), a potent greenhouse gas, can be produced through a variety of biochemical pathways. Nitrifier denitrification, i.e. nitrite reduction by ammonia oxidizers, is one of them. The existence of this pathway has been identified in monoculture studies, and it is increasingly suggested that it may contribute substantially to N2O production in soil. However, methodological drawbacks thus far prohibited conclusive proof of its occurrence in soil-based studies. As soils constitute the major global source of N2O, understanding the significance of nitrifier denitrification is indispensable to effective mitigation of N2O emissions. We developed and applied a new approach to identify the presence of nitrifier denitrification in soil. N2O production was studied in soil incubations using oxygen (O) and nitrogen (N) isotope enrichment tracing, while accounting for O exchange between water and intermediate products of the N transformations. Twelve soils were studied which covered a diversity of soil and land-use types across Europe. We additionally aimed to link variation in N2O production pathways to differences in the microbial community by examining microbial biomass C and N and phospholipid fatty acid (PLFA) patterns. Our results showed that in at least five of the soils studied, nitrifier denitrification must have contributed to total N2O production. Data revealed that in fact it may have been responsible for all NH4+-derived N2O in most soils. In contrast, N2O as by-product of ammonia oxidation contributed marginally, if any, to total production. Surprisingly, none of the examined microbial parameters could adequately explain differences in N2O production. Soil pH was the best predictor of the observed relative contributions of the pathways. We conclude that with our approach we finally demonstrate that nitrifier denitrification can indeed occur in soil, and may in fact be responsible for the majority of total nitrifier-induced N2O production. Our data further suggest that microbial community composition may not be a key driver of in the relative significance of different N2O production pathways, and that pH may be a principal factor determining nitrifier-induced N2O production.

Kool, D. M.; Wrage, N.; Zechmeister-Boltenstern, S.; Pfeffer, M.; Brus, D.; Oenema, O.; van Groenigen, J.



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)



Combining Computational Prediction of Cis-Regulatory Elements with a New Enhancer Assay to Efficiently Label Neuronal Structures in the Medaka Fish  

PubMed Central

The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates.

Bourrat, Franck; Gruhl, Franziska; Dewar, Ken; Blanchette, Mathieu; Wittbrodt, Joachim; Ettwiller, Laurence



Label Claims  

Center for Food Safety and Applied Nutrition (CFSAN)

... Print; Share; E-mail. Home; Food; Ingredients, Packaging & Labeling; Labeling & Nutrition. ... that petitions and notifications be submitted in electronic form ... More results from


Difference in Mass Analysis Using Labeled Lysines (DIMAL-K): A New, Efficient Proteomic Quantification Method Applied to the Analysis of Astrocytic Secretomes  

Microsoft Academic Search

Here we describe an original strategy for unbiased quan- tification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely

Nicolas Delcourt; Patrick Jouin; Joel Poncet; Emmanuelle Demey; Eric Mauger; Joel Bockaert; Philippe Marin; Nathalie Galeotti



Efficient radiosynthesis of carbon-11 labelled uncharged Thioflavin T derivatives using [ 11C]methyl triflate for ? -amyloid imaging in Alzheimer's Disease with PET  

Microsoft Academic Search

The synthesis of carbon-11 amino function labelled uncharged Thioflavin T derivatives is known to be performed by reaction of the demethyl-precursors with [11C]methyl iodide but the labelling yields are only mediocre. The use of [11C]methyl triflate improved the radiochemical yield of three potential ?-amyloid imaging PET-radiotracers significantly. Performance of the labelling reaction by reacting the corresponding precursor molecules with [11C]methyl

C. Solbach; M. Uebele; G. Reischl; H.-J. Machulla



Efficient \\  

Microsoft Academic Search

In 1999, Poupard and Stern proposed on the fly signature scheme (PS-scheme), which aims at minimizing the on-line computa- tional work for a signer. In this paper, we propose more efficient on the fly signature schemes by improving the PS-scheme. In PS-scheme, the size of secret-key is fixed by modulus n, so that this feature leads to some drawbacks in

Takeshi Okamoto; Mitsuru Tada; Atsuko Miyaji



Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures  

SciTech Connect

Many cellular processes are regulated by reversible protein phosphorylation and the ability to identify and quantify phosphoproteins from proteomes is essential for gaining a better understanding of these dynamic cellular processes. However, 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 abundance 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 laboratory1-2, where the O-phosphate moiety on phosphoseryl or phosphothreonyl residues were derivatized by hydroxide ion-medated B-elimination followed by the 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 LC-MS/MS. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures was demonstrated using casein phosphoproteins. Its utility for proteomic applications is demonstrated by the labeling of soluble proteins from human breast cancer cell line.

Qian, Weijun (BATTELLE (PACIFIC NW LAB)); Goshe, Michael B. (North Carolina State University); Camp, David G. (BATTELLE (PACIFIC NW LAB)); Yu, Li-Rong (BATTELLE (PACIFIC NW LAB)); Tang, Keqi (BATTELLE (PACIFIC NW LAB)); Smith, Richard D. (BATTELLE (PACIFIC NW LAB))



Comparison of seasonal variations in water-use efficiency calculated from the carbon isotope composition of tree rings and flux data in a temperate forest.  


Tree-ring ?(13) C is often interpreted in terms of intrinsic water-use efficiency (WUE) using a carbon isotope discrimination model established at the leaf level. We examined whether intra-ring ?(13) C could be used to assess variations in intrinsic WUE (W(g), the ratio of carbon assimilation and stomatal conductance to water) and variations in ecosystem WUE (W(t) , the ratio of C assimilation and transpiration) at a seasonal scale. Intra-ring ?(13) C was measured in 30- to 60-µm-thick slices in eight oak trees (Quercus petraea). Canopy W(g) was simulated using a physiologically process-based model. High between-tree variability was observed in the seasonal variations of intra-ring ?(13) C. Six trees showed significant positive correlations between W(g) calculated from intra-ring ?(13) C and canopy W(g) averaged over several days during latewood formation. These results suggest that latewood is a seasonal recorder of W(g) trends, with a temporal lag corresponding to the mixing time of sugars in the phloem. These six trees also showed significant negative correlations between photosynthetic discrimination ? calculated from intra-ring ?(13) C, and ecosystem W(t), during latewood formation. Despite the observed between-tree variability, these results indicate that intra-ring ?(13) C can be used to access seasonal variations in past W(t). PMID:20955221

Michelot, Alice; Eglin, Thomas; Dufrêne, Eric; Lelarge-Trouverie, Caroline; Damesin, Claire



18O labeling over a coffee break: a rapid strategy for quantitative proteomics.  


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, (18)O 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



Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion  

Microsoft Academic Search

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental 13C\\/15N\\/2H isotope-labeled single- stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments.

Frank H. T. Nelissen; Frederic C. Girard; Marco Tessari; Hans A. Heus; Sybren S. Wijmenga



Considerations in the Development of the Utility of Stable Isotopes in Science, Medicine, and Agriculture, and Environmental Studies.  

National Technical Information Service (NTIS)

The prospects for the broad scale development of the utility of stable isotopes in science, medicine, agriculture, and environmental studies are considered with emphasis on the current status of isotope production, synthesis of isotopically labelled compo...

N. A. Matwiyoff



Stable isotope coded derivatizing reagents as internal standards in metabolite profiling.  


Gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometric (MS) detection have become the two main techniques for the analysis of metabolite pools (i.e. Metabolomics). These technologies are especially suited for Metabolite Profiling analysis of various metabolite groups due to high separation capabilities of the chromatographs and high sensitivity of the mass analysers. The trend in quantitative Metabolite Profiling is to add more metabolites and metabolite groups in a single method. This should not be done by compromising the analytical precision. Mass spectrometric detection comes with certain limitations, especially in the quantitative aspects as standards are needed for conversion of ion abundance to concentration and ionization efficiencies are directly dependent on eluent conditions. This calls for novel strategies to counteract all variables that can influence the quantitative precision. Usually, internal standards are used to correct any technical variation. For quantitation of single or just a few analytes this can be executed with spiking isotopically labeled standards. However, for more comprehensive analytical tasks, e.g. profiling tens or hundreds of analytes simultaneously, this strategy becomes expensive and in many cases isotopically labeled standards are not available. An alternative is to introduce a derivatizing step where the sample is derivatized with naturally labeled reagent, while a standard solution is separately derivatized with isotopically labeled reagent and spiked into the sample solution prior to analysis. This strategy, named isotope coded derivatization - ICD, is attractive in the emerging field of quantitative Metabolite Profiling where current protocols can easily comprise over hundred metabolites. This review provides an overview of isotopically labeled derivatizing reagents that have been developed for important metabolite groups with the aim to improve analytical performance and precision. PMID:23628173

Bruheim, Per; Kvitvang, Hans Fredrik Nyvold; Villas-Boas, Silas G



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


High-efficiency astatination of antibodies using N-iodosuccinimide as the oxidising agent in labelling of N-succinimidyl 3-(trimethylstannyl)benzoate  

Microsoft Academic Search

Monoclonal antibodies C215, reactive with colorectal carcinomas, and MOv18, reactive with most of the ovarian carcinomas, were radiohalogenated with [211At]astatine. The radiohalogen was conjugate coupled to antibodies via the intermediate labelling reagent N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE) in a two-step, single-pot reaction. Optimisation of the labelling of the reagent was achieved using N-iodosuccinimide, NIS, as the oxidising agent. The yields ranged from 69–95%

S. Lindegren; H. Andersson; T. Bäck; L. Jacobsson; B. Karlsson; G. Skarnemark



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



Rare Isotope Accelerators  

NASA Astrophysics Data System (ADS)

The next frontier for low-energy nuclear physics involves experimentation with accelerated beams of short-lived radioactive isotopes. A new facility, the Rare Isotope Accelerator (RIA), is proposed to produce large amount of these rare isotopes and post-accelerate them to energies relevant for studies in nuclear physics, astrophysics and the study of fundamental interactions at low energy. The basic science motivation for this facility will be introduced. The general facility layout, from the 400 kW heavy-ion superconducting linac used for production of the required isotopes to the novel production and extraction schemes and the highly efficient post-accelerator, will be presented. Special emphasis will be put on a number of technical breakthroughs and recent R&D results that enable this new facility.

Savard, Guy



Simplified and efficient labeling of human platelets in plasma using indium-111-2-mercaptopyridine-N-oxide: preparation and evaluation  

SciTech Connect

A water soluble sodium salt of 2-mercaptopyridine-N-oxide (Merc) was evaluated that quantitatively chelated /sup 111/In at a pH range 0.7 to 7.4, and allowed greater than 80% incorporation of /sup 111/In in platelets in plasma. This was dependent on pH, Merc concentration, and platelet concentration. Platelets were labeled using either preformed (/sup 111/In)Merc or incubating platelets with 2.5 dry Merc first and then with /sup 111/In. The latter method provided a simple kit procedure that labeled platelets with negligible alteration of in vitro aggregability. In dogs, labeled platelets had normal survival (7.5 days), 66% recovery, detected vascular thrombi, and pulmonary emboli by scintigraphy. In the kit procedure, the use of Merc compared favorably to that of oxine and tropolone.

Thakur, M.L.; McKenney, S.L.; Park, C.H.



The Elusive Structure of Pd2(dba)3. Examination by Isotopic Labeling, NMR Spectroscopy, and X-ray Diffraction Analysis: Synthesis and Characterization of Pd2(dba-Z)3 Complexes.  


Pd(0)2(dba)3 (dba = E,E-dibenzylidene acetone) is the most widely used Pd(0) source in Pd-mediated transformations. Pd(0)2(dba-Z)3 (Z = dba aryl substituents) complexes exhibit remarkable and differential catalytic performance in an eclectic array of cross-coupling reactions. The precise structure of these types of complexes has been confounding, since early studies in 1970s to the present day. In this study the solution and solid-state structures of Pd(0)2(dba)3 and Pd(0)2(dba-Z)3 have been determined. Isotopic labeling ((2)H and (13)C) has allowed the solution structures of the freely exchanging major and minor isomers of Pd(0)2(dba)3 to be determined at high field (700 MHz). DFT calculations support the experimentally determined major and minor isomeric structures, which show that the major isomer of Pd(0)2(dba)3 possesses bridging dba ligands found exclusively in a s-cis,s-trans conformation. For the minor isomer one of the dba ligands is found exclusively in a s-trans,s-trans conformation. Single crystal X-ray diffraction analysis of Pd(0)2(dba)3·CHCl3 (high-quality data) shows that all three dba ligands are found over two positions. NMR spectroscopic analysis of Pd(0)2(dba-Z)3 reveals that the aryl substituent has a profound effect on the rate of Pd-olefin exchange and the global stability of the complexes in solution. Complexes containing the aryl substituents, 4-CF3, 4-F, 4-t-Bu, 4-hexoxy, 4-OMe, exhibit well-resolved (1)H NMR spectra at 298 K, whereas those containing 3,5-OMe and 3,4,5-OMe exhibit broad spectra. The solid-state structures of three Pd(0)2(dba-Z)3 complexes (4-F, 4-OMe, 3,5-OMe) have been determined by single crystal X-ray diffraction methods, which have been compared with Goodson's X-ray structure of Pd(0)2(dba-4-OH)3. PMID:23701049

Kapdi, Anant R; Whitwood, Adrian C; Williamson, David C; Lynam, Jason M; Burns, Michael J; Williams, Thomas J; Reay, Alan J; Holmes, Jordan; Fairlamb, Ian J S



Incorporating concentration dependence in stable isotope mixing models  

Microsoft Academic Search

Stable isotopes are often used as natural labels to quantify the contributions of multiple sources to a mixture. For example, C and N isotopic signatures can be used to determine the fraction of three food sources in a consumer's diet. The standard dual isotope, three source linear mixing model assumes that the proportional contribution of a source to a mixture

Donald L. Phillips; Paul L. Koch




EPA Science Inventory

Stable isotopes are often used as natural labels to quantify the contributions of multiple sources to a mixture. For example, C and N isotopic signatures can be used to determine the fraction of three food sources in a consumer's diet. The standard dual isotope, three source li...


High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.  


The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows. PMID:23033477

Thiede, Bernd; Koehler, Christian J; Strozynski, Margarita; Treumann, Achim; Stein, Robert; Zimny-Arndt, Ursula; Schmid, Monika; Jungblut, Peter R



Plasma Biomarker Discovery Using 3D Protein Profiling Coupled with Label-Free Quantitation  

PubMed Central

In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC–MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC–MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as “apparent” biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC–MS/MS with label-free quantitation of candidate biomarkers.

Beer, Lynn A.; Tang, Hsin-Yao; Barnhart, Kurt T.; Speicher, David W.



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.



Convenient and Efficient Synthesis of a Lanthanide3+-Coordinated, Diethylene Triamine Pentaacetic Acid Labeled Biopolymer as an Assay for the Cholecystokinin B Receptor  

PubMed Central

To develop an assay for the cholecystokinin B receptor with an Eu3+-labeled cholecystokinin peptide via a diethylene triamine pentaacetic acid chelating linker, a commercial dianhydride diethylene triamine pentaacetic acid precursor was directly attached to the N-terminus of cholecystokinin peptides by a solid-phase synthesis method with a satisfactory yield and purity after reverse-phase high-performance liquid chromatography separation. Lanthanide was then coordinated to the peptide via a diethylene triamine pentaacetic acid bifunctional agent. This method is a useful approach to the large-scale synthesis of lanthanide3+-coordinated, diethylene triamine pentaacetic acid labeled biopolymers. This research provides not only a simple and convenient method for the preparation of lanthanide-based peptide ligand libraries but also possible lanthanide-based high-throughput screening of peptide receptors with a timeresolved fluorescence assay system. Five biopolymers were synthesized and characterized with high-resolution electrospray ionization in this study.

Gao, F.; Handl, H.; Vagner, J.; Hruby, V.; Gillies, R.



Point set labeling with sliding labels  

Microsoft Academic Search

This paper discusses algorithms for labeling sets of points in the plane, where labels are not restricted to some finite num- ber of positions. We show that continuously sliding labels allows more points to be labeled both in theory and in prac- tice. We define six different models of labeling, and analyze how much better—more points get a label—one model

Marc J. van Kreveld; Tycho Strijk; Alexander Wolff



Studies on the Transfer of Myeloma Protein Synthesis with RNA Isolated from the C3h Plasma Cell Tumor (X5563). Ii. Uptake of Isotopically Labeled Tumor RNA by Lymphoid Cells.  

National Technical Information Service (NTIS)

Studies were carried out to establish the conditions necessary for increasing quantitatively the uptake of exogenous RNA by incubating dissociated lymphoid cells of C3H mice with H3-or P32-labeled RNA in vitro. The problem of adsorption of labeled RNA ont...

P. H. Chin M. S. Silverman



A Simple Procedure for Effective Quenching of Trypsin Activity and Prevention of 18O-Labeling Back-Exchange  

PubMed Central

Trypsin-catalyzed stable isotope 16O/18O-labeling of the C-terminal carboxyl groups of peptides is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by residual trypsin that can catalyze the back-exchange of 18O with 16O after labeling. Here, we demonstrate through a detailed evaluation that boiling the peptide sample for 10 minutes provides a simple means for completely quenching residual trypsin activity and preventing oxygen back-exchange in 18O-labeled samples. We also observed that the presence of organic solvents such as acetonitrile made quenching trypsin activity less efficient. Finally, current 18O-labeling methods that typically employ immobilized trypsin result in significant sample losses due to non-specific binding of peptides on the resin, making their application toward smaller biological samples increasingly impractical. We present here an improved 18O-labeling protocol that is more applicable to microscale biological samples by using solution-phase trypsin instead of immobilized trypsin to overcome the non-specific sample loss issue encountered with the use of immobilized trypsin. The ability to generate stably 18O-labeled samples without back-exchange should enable more effective applications of 18O-labeled toward large-scale biomarker discovery and validations where an 18O-labeled sample can be used as a common reference for quantitation.

Petritis, Brianne O.; Qian, Wei-Jun; Camp, David G.; Smith, Richard D.



An efficient protocol for the purification and labeling of entire yeast septin rods from E.coli for quantitative in vitro experimentation  

PubMed Central

Background The detailed understanding of the functions and mechanisms of the actin and microtubuli cytoskeleton depended, besides innovative methods in live cell imaging, on the purification and labeling of its constituents. This allowed researchers to quantitatively measure filament stability, the rates of filament turnover as well as the determination of the influence of cofactors on filament formation and structure. Septins form the least understood class of cytoskeletal structures in nearly all eukaryotic cells so far examined. In yeast, they comprise a family of proteins (Cdc3, Cdc10, Cdc11, Cdc12, Shs1) that form a co-polymeric, ring-like structure beneath the membrane. This ring serves as a template for the formation of a new bud neck and as a landing pat for proteins involved in polar growth and cytokinesis. Further progress in investigating the mechanisms of septin-structure formation and regulation is hampered by the lack of protocols to modify homogenous samples of purified septins with useful probes for in vitro biochemical studies. Results We present a protocol for the purification and labeling of yeast septin rods. The four individual septin subunits were co-expressed in E.coli. One subunit of the septin polymer was expressed as SNAP tag fusion protein allowing for rapid and stoichiometric labeling with derivatized Benzylguanine (BG). To demonstrate the applicability of our approach, we introduced two different SNAP tag substrates: septin rods labeled with fluorescent BG compounds enabled us to monitor the formation of filaments by fluorescence microscopy whereas BG-biotin was used to couple septin rods to a sensor chip for quantitative surface plasmon resonance binding experiments. In a first application, we determined the affinity and the binding kinetics of the yeast protein Bni5 to the individually coupled septin rods. In a further application we could demonstrate that a once formed septin rod hardly exchange its subunits. Conclusions The herein introduced protocol of purifying SNAP tag modified septins from E.coli allowed us to derivatize the obtained septin rods with probes for the further in vitro characterization of this class of cytoskeletal elements. The availability of a very diverse set of SNAP tag substrates should open the way to investigate different aspects of septin biochemistry in mechanistic detail.



Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: Symmetry of the carbonyl interactions and close equivalence of the Q{sub B} vibrations in Rhodobacter sphaeroides and Rhodopseudomonas viridis probed by isotope labeling  

SciTech Connect

The photoreduction of the secondary quinone acceptor Q{sub B} in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis has been investigated by light-induced FTIR difference spectroscopy of RCs reconstituted with several isotopically labeled ubiquinones. The labels used were {sup 18}O on both carbonyls and {sup 13}C either uniformly or selectively at the 1- or the 4-position, i.e., on either one of the two carbonyls. The Q{sub B}{sup {minus}}/Q{sub B} spectra of RCs reconstituted with the isotopically labeled and unlabeled quinones as well as the double differences calculated form these spectra exhibit distinct isotopic shifts for a numer of bands attributed to vibrations of Q{sub B} and Q{sub B}{sup {minus}}. The vibrational modes of the quinone in the Q{sub B} site are compared to those of ubiquinone in vitro, leading to band assignments for the C{double_bond}O and C{double_bond}C vibrations of the neutral Q{sub B} and for the C---O and C---C of the semiquinone. The C{double_bond}O frequency of each of the carbonyls of the unlabeled quinone is revealed at 1641 cm{sup {minus}1} for both species. This demonstrates symmetrical and weak hydrogen bonding of the two C{double_bond}O groups to the protein at the Q{sub B} site. In contrast, the C{double_bond}C vibrations are not equivalent for selective labeling at C{sub 1} or at C{sub 4}, although they both contribute to the {approximately}1611-cm{sup {minus}1} band in the Q{sub B}{sup {minus}}/Q{sub B} spectra of the two species. Compared to the vibrations of isolated ubiquinone, the C{double_bond}C mode of Q{sub B} does not involve displacement of the C{sub 4} carbon atom, while the motion of C{sub 1} is not hindered. Further analysis of the spectra suggests that the protein at the binding site imposes a specific constraint on the methoxy and/or the methyl group proximal to the C{sub 4} carbonyl. 49 refs., 5 figs.

Breton, J.; Berger, G.; Nabedryk, E. [SBE/DBCM and SMM/DBCM, CEA-Saclay (France)] [and others



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)



Application of the natural isotopes in researches of water resources (in Macedonia).  

National Technical Information Service (NTIS)

Isotopes techniques based on the natural stable isotopes are efficient hydrological tool for the determination of the origin of springs and other waters. Through practical example, this paper presents the application of stabile isotopes deuterium (H-2) an...

T. Anovski B. Andonovski N. Jovanovski



Improvement of multi jet low pressure impactor for high collection efficiency of UF5 in the molecular laser isotope separation of uranium  

Microsoft Academic Search

A numerical and experimental study for the collection of photo-produced UF5 particles was performed for the low pressure impactors which have different design factors at typical flow conditions (upstream pressure of the impactor = 10–15 Torr, pressure ratio of downstream to upstream of the impactor, PdownPup = 0.2–0.5) in the molecular laser isotope separation of uranium at RIKEN (RIMLIS). Smaller

Yoshikazu Kuga; Benjamin Jurcik; Sakae Satooka; Kazuo Takeuchi



Effects of Soil Strength on the Relation of Water-Use Efficiency and Growth to Carbon Isotope Discrimination in Wheat Seedlings  

PubMed Central

The ratio of carbon accumulation to transpiration, W, of wheat (Triticum aestivum L.) seedlings increased with increasing soil strength, measured as soil penetrometer resistance, and this was already apparent at the two leaf stage. The ratio was negatively correlated with carbon isotope discrimination, in accord with theory. This means that decrease in intercellular partial pressure of CO2 accounted for an important part of the increase in W with increasing soil strength. Despite a lower CO2 concentration in the leaves at high soil strength, assimilation rate per unit leaf area was enhanced. Greater ribulose 1,5-bisphosphate carboxylase activity confirmed that photosynthetic capacity was actually increased. This pattern of opposite variation of assimilation rate and of stomatal conductance is unusual. The ratio of plant carbon mass to leaf area increased markedly with increasing soil strength, mainly because of a greater investment of carbon into roots than into shoots. A strong negative correlation was found between this ratio and carbon isotope discrimination. For a given increase in discrimination, decrease in carbon mass per leaf area was proportionally larger than decrease in assimilation rate, so that relative growth rate was positively correlated to carbon isotope discrimination.

Masle, Josette; Farquhar, Graham D.



Selective Isotope Determination of Uranium using HR-RIMS  

SciTech Connect

The detection of lowest abundances of the ultra trace isotope {sup 236}U in environmental samples requires an efficient detection method which allows a high elemental and isotopic selectivity to suppress neighbouring isotopes of the same element and other background. High Resolution Laser Resonance Ionization Mass Spectrometry (HR-RIMS) uses the individual electron structure of each isotope to provide an outstanding element and isotope selective ionization.

Raeder, S.; Fies, S.; Wendt, K. D. A. [Institut fuer Physik, Johannes Gutenberg Universitaet Mainz, 55128 Mainz (Germany); Tomita, H. [Nagoya University (Japan)



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.



Stable isotope dilution assays in mycotoxin analysis.  


The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis. PMID:18060393

Rychlik, Michael; Asam, Stefan



Failure to label baboon milk intrinsically with iron  

SciTech Connect

The widely held belief that 50% of the iron in human milk is absorbed is based on studies that have used an extrinsic radioactive iron tag. To determine the validity of an extrinsic tag, it is necessary to label the milk intrinsically with one isotope and to compare absorption of this isotope with absorption of another isotope added as the extrinsic tag. We chose the baboon as a model and infused 59Fe intravenously. In each of three attempts we failed to label the milk intrinsically.

Figueroa-Colon, R.; Elwell, J.H.; Jackson, E.; Osborne, J.W.; Fomon, S.J. (Univ. of Iowa, Iowa City (USA))



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)



Pulsed 86Sr-labeling and NanoSIMS imaging to study coral biomineralization at ultra-structural length scales  

NASA Astrophysics Data System (ADS)

A method to label marine biocarbonates is developed based on a concentration enrichment of a minor stable isotope of a trace element that is a natural component of seawater, resulting in the formation of biocarbonate with corresponding isotopic enrichments. This biocarbonate is subsequently imaged with a NanoSIMS ion microprobe to visualize the locations of the isotopic marker on sub-micrometric length scales, permitting resolution of all ultra-structural details. In this study, a scleractinian coral, Pocillopora damicornis, was labeled 3 times with 86Sr-enhanced seawater for a period of 48 h with 5 days under normal seawater conditions separating each labeling event. Two non-specific cellular stress biomarkers, glutathione-S-transferase activity and porphyrin concentration plus carbonic anhydrase, an enzymatic marker involved in the physiology of carbonate biomineralization, as well as unchanged levels of zooxanthellae photosynthesis efficiency indicate that coral physiological processes are not affected by the 86Sr-enhancement. NanoSIMS images of the 86Sr/44Ca ratio in skeleton formed during the experiment allow for a determination of the average extension rate of the two major ultra-structural components of the coral skeleton: Rapid Accretion Deposits are found to form on average about 4.5 times faster than Thickening Deposits. The method opens up new horizons in the study of biocarbonate formation because it holds the potential to observe growth of calcareous structures such as skeletons, shells, tests, spines formed by a wide range of organisms under essentially unperturbed physiological conditions.

Brahmi, C.; Domart-Coulon, I.; Rougée, L.; Pyle, D. G.; Stolarski, J.; Mahoney, J. J.; Richmond, R. H.; Ostrander, G. K.; Meibom, A.



Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization.  


Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities. PMID:21824243

Wang, Shanquan; He, Jianzhong



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



A Prime Number Labeling Scheme for Dynamic Ordered XML Trees  

Microsoft Academic Search

Efficient evaluation of XML queries requires the determination of whether a relationship exists between two elements. A number of labeling schemes have been designed to label the element nodes such that the relationships between nodes can be easily determined by comparing their labels. With the increased popularity of XML on the Web, finding a labeling scheme that is able to

Xiaodong Wu; Mong-li Lee; Wynne Hsu



Biomedical Research Applications of Electromagnetically Separated Enriched Stable Isotopes.  

National Technical Information Service (NTIS)

The current and projected annual requirements through 1985 for stable isotopes enriched by electromagnetic separation methods were reviewed for applications in various types of biomedical research: (1) medical radioisotope production, labeled compounds, a...

R. M. Lambrecht



Semiconductor isotope engineering  

SciTech Connect

Isotopic control of semiconductor crystals offers a wide range of scientific and technical opportunities. We review neutron transmutation doping of natural and isotopically controlled semiconductor structures, special properties of isotope superlattices, the effect of host isotopes on local vibrational modes of low mass impurities, and intrinsic properties which depend on isotope mass and isotopic composition of single crystals.

Haller, E.E.



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.



Technical Issues for Building-Energy-Use Labels.  

National Technical Information Service (NTIS)

Current market practice in building energy efficiency lags 15 to 30 years behind current cost-effective conservation practice, in part due to lack of credible information about home energy efficiency. It is shown that building energy efficiency labels are...

A. H. Rosenfeld B. S. Wagner



Photodisintegration of Lithium Isotopes  

NASA Astrophysics Data System (ADS)

It is clear that new experimental data is needed to compare with recent Lorentz Integral Transform calculations of the photodisintegration cross sections of the lithium isotopes. We describe a recent measurement on these isotopes performed with the monochromatic, polarized photon beam at the High Intensity Gamma-Ray Source (HIGS) at Duke University in Durham, NC, USA. The Blowfish Neutron Detector Array, a segmented neutron detector array with good angular resolution that covers .5ex1 -.1em/ -.15em.25ex4 of 4? steradians, was used to detect photoneutrons. Clear separation of various reaction channels is possible which allows detector efficiencies to be accurately modeled using a GEANT4 simulation. Several methods for obtaining the incident photon flux are available so precision cross sections between 8 and 35 MeV can be obtained. )

Pywell, R. E.; Wurtz, W. A.; Norum, B.; Kucucker, S.; Sawatzky, B. D.; Weller, H. R.; Ahmed, M. A.; Stave, S.



A label-free mass spectrometry method for the quantification of protein isotypes  

Microsoft Academic Search

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for

Robert D. Winefield; Todd D. Williams; Richard H. Himes



Insights from Stable Isotopes on the Role of Terrestrial Ecosystems in the Global Carbon Cycle  

Microsoft Academic Search

The use of isotopic tracers in organic matter, water, and atmospheric gases has become an important component of the study of ecology and global change. Physiological and physical processes discriminate against heavy isotopes in predictable ways, so that measurements of isotopes at natural abundance, i.e., naturally occurring concentrations as opposed to artificial labeling experiments, can provide useful information about biological

Diane E. Pataki; Chun-Ta Lai; Charles D. Keeling; James R. Ehleringer


Site-specific labeling of supercoiled DNA  

Microsoft Academic Search

Visualization of site-specific labels in long linear or circular DNA allows unambiguous identification of various local DNA structures. Here we describe a novel and efficient approach to site-specific DNA labeling. The restriction enzyme SfiI binds to DNA but leaves it intact in the presence of calcium and therefore may serve as a protein label of 13 bp recognition sites. Since

Alexander Y. Lushnikov; Vladimir N. Potaman; Yuri L. Lyubchenko



Utraviolet-B effects on stomatal density, water use efficiency, and stable carbon isotope discrimination in four glasshouse-grown soybean cultivars  

Technology Transfer Automated Retrieval System (TEKTRAN)

Interactions between UV-B radiation and drought stress have been observed but the underlying mechanisms have not been thoroughly investigated. We hypothesized that UV-B radiation would improve water use efficiency by its effects on epidermal development, specifically stomatal density and leaf gas e...


Ultraviolet-B effects on stomatal density, water-use efficiency, and stable carbon isotope discrimination in four glasshouse-grown soybean (Glyicine max) cultivars  

Technology Transfer Automated Retrieval System (TEKTRAN)

Interactions between UV-B radiation and drought stress have been studied but the underlying mechanisms have not been thoroughly investigated. We hypothesized that ambient UV-B radiation would improve water-use efficiency (WUE) by its effects on epidermal development, specifically reduced stomatal de...


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))



A metathesis route for BODIPY labeled polyolefins.  


It is demonstrated how acyclic diene metathesis polymerization (ADMET) provides an efficient strategy for the labeling of polyolefins. The versatility of phosphorus chemistry allows designing substituted BODIPY monomers or chain stoppers for the synthesis of precise labeled (degradable) polyphosphoesters. PMID:23869751

Marsico, Filippo; Turshatov, Andrey; Weber, Katja; Wurm, Frederik R



Stable Isotope Dilution for Hazardous Waste Incineration.  

National Technical Information Service (NTIS)

The report gives results of a project to determine if a proposed catalytic exchange procedure could be adapted to produce the labeled analog materials necessary for isotope dilution gas chromatography/mass spectrometry (GC/MS) analysis. It is related to a...

P. W. Ryan



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



Ultraviolet-B effects on stomatal density, water-use efficiency, and stable carbon isotope discrimination in four glasshouse-grown soybean ( Glyicine max) cultivars  

Microsoft Academic Search

Interactions between UV-B radiation and drought stress have been studied but the underlying mechanisms have not been thoroughly investigated. We hypothesized that UV-B radiation would improve water-use efficiency (WUE) by its effects on epidermal development, specifically stomatal density, and leaf gas exchange. Four lines of soybean (Glycine max: Essex, Williams, OX921, and OX922) were grown for 28 days in a

Dennis C. Gitz; Lan Liu-Gitz; Steven J. Britz; Joe H. Sullivan



Production of carbon isotopes by laser separation  

NASA Astrophysics Data System (ADS)

Since the advent of lasers, these unique sources of highly intense and monochromatic radiation have been proposed as excellent tools to induce or catalyze chemical reactions. Due to the great interest to the problem of isotope production, investigation and application, the laser method of isotope separation has received the most attention worldwide and may be the first major commercial application of lasers to chemistry. Laser methods of isotope separation are based on high selectivity and power of laser sources of radiation. One of the most prominent method is based on the effect is isotope-selective multiphoton dissociation of molecules by IR-radiation (MLIS-method). This phenomena was discovered in Russia in 1974 and developed from scientific investigations to industrial scale production of 13C isotopes in collaboration between the Kurchatov Institute of Atomic Energy, TRINITI and Institute of Spectroscopy of RAS. Demonstration facilities for sulfur and carbon isotope separation with average productivity up to 2 g/h have been created as a result of collaboration and these systems are aimed at optimization of MLIS process and evaluation of its cost efficiency. Experiments show that laser produced isotopes are far cheaper as compared to any conventional technique. Results of basic scientific research, existing technological cooperation allow to start building a laser isotope separation plant. Light element isotopes produced there can answer a wide variety of demands in many technologies. These isotopes can be readily used in medicine, agriculture, environmental monitoring, etc.

Baranov, Vladimir Y.; Dyad'kin, A. P.; Maluta, D. D.; Kuzmenko, V. A.; Pigulskiy, S. V.; Mezhevov, Vladimir S.; Letokhov, Vladilen S.; Laptev, V. B.; Ryabov, E. A.; Yarovoi, I. V.; Zarin, V. B.; Podoryashy, A. S.



RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny  

Microsoft Academic Search

accountable for it. In this study stable-isotope-labeled (13C)phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this

Mike Manefield; Andrew S. Whiteley; Robert I. Griffiths; Mark J. Bailey



Synthesis and pharmacokinetics of thiacoccide labeled with tritium  

SciTech Connect

To obtain information on the pharmacokinetics of thiacoccide, the authors effect the synthesis of an analog of thiacoccide, viz., the hydrochloride of 2-methyl-N-(2'-n-propyl-4'-aminopyrimid-5'-ylmethyl)pyridinium chloride (I) labeled with tritium. The simplest way to introduce a tritium label into (I) is by isotopic exchange of hydrogen of (I) with H/sup 3/HO. Typical material obtained from isotopic exchange with water labeled with tritium is shown. The dynamics of /sup 3/H distribution in the organism of the chick and the rate of its elimination on single administration of thiacoccide containing isotope in both the methyl group and in the pyridine ring are shown.

Shestakov, A.D.; Kaminskii, Yu.L.; Nurova, I.M.; Nikitina, V.N.; Zaionts, V.I.; Maksimova, O.V.; Mikhailov, G.A.



Approximating Labeled Markov Processes  

Microsoft Academic Search

We study approximate reasoning about continuous-state labeled Markov processes. We show how to approximate a labeled Markov process by a family offinite-state labeled Markov chains. We show that the collection of labeled Markov processes carries a Polish space structure with a countable basis given by finite state Markov chains with ra- tional probabilities. The primary technical tools that we develop

Josee Desharnais; Vineet Gupta; Radha Jagadeesan; Prakash Panangaden



Approximating labelled Markov processes  

Microsoft Academic Search

Labelled Markov processes are probabilistic versions of labelled transition systems. In general, the state space of a labelled Markov process may be a continuum. In this paper, we study approximation techniques for continuous-state labelled Markov processes.We show that the collection of labelled Markov processes carries a Polish-space structure with a countable basis given by finite-state Markov chains with rational probabilities;

Josee Desharnais; Vineet Gupta; Radha Jagadeesan; Prakash Panangaden



Quantitative determination of oxidative base damage in DNA by stable isotope-dilution mass spectrometry.  


For understanding of the role of oxidative DNA damage in biological processes such as mutagenesis and carcinogenesis, it is essential to identify and quantify this type of DNA damage in cells. This can be achieved by gas chromatography/mass spectrometry. The present study describes the quantification of modified bases in DNA by isotope-dilution mass spectrometry with the use of stable isotope-labeled analogues as internal standards. A number of isotopically labeled DNA bases were synthesized. The mass spectra of their trimethylsilyl derivatives were recorded. Calibration plots were obtained for known quantities of modified bases and their isotope-labeled analogues. Quantification of various modified DNA bases by isotope-dilution mass spectrometry was demonstrated in isolated chromatin exposed to ionizing radiation. The results indicate that gas chromatography/stable isotope-dilution mass spectrometry is an ideally suited technique for selective and sensitive quantification of modified bases in DNA. PMID:8416801

Dizdaroglu, M



Antibody-dependent antitumor cytotoxicity by human monocytes cultured with recombinant macrophage colony-stimulating factor. Induction of efficient antibody-mediated antitumor cytotoxicity not detected by isotope release assays  

PubMed Central

Macrophage colony-stimulating factor (M-CSF) is known to stimulate proliferation of monocyte/macrophage progenitors and enhance in vitro antitumor cytotoxicity by murine macrophages. In this paper we have shown that recombinant human M-CSF causes human peripheral blood monocytes to differentiate in culture into metabolically active macrophage-like cells. These cells mediate very efficient antibody- dependent cellular cytotoxicity (ADCC) against human melanoma and neuroblastoma cell lines in the presence of two murine IgG3 mAbs (3F8 and R24). They also mediate antibody-independent cytotoxicity (or cytostasis) to a lesser extent. Human serum had an inconsistent effect on ADCC, but often induced similar high levels of ADCC. Cytotoxicity was measured using a novel ELISA to detect surviving tumor cells after ADCC. Two conventional isotope-release assays (51Cr and [3H]TdR) underestimated or entirely failed to detect ADCC by M-CSF-activated monocytes. Optimal activation occurred with 100-300 U/ml of M-CSF, and required 9-11 d for completion. Most of the M-CSF cultured monocytes expressed the low-affinity Fc receptor (CD16). ADCC by cells of the monocyte/macrophage lineage using murine IgG3 mAbs may have significance for the immunotherapy of human malignancies.



Metabolic isotopomer labeling systems. Part III: path tracing.  


Isotope labeling systems (ILSs) are sets of balance equations that quantitatively describe the distribution of isotopic tracers in metabolic networks. The solution of ILSs, i.e., the calculation of isotopic labeling distributions (mostly in steady state) is the fundamental computational step of (13)C metabolic flux analysis (MFA). Aiming at a deeper analytical understanding of ILSs a new approach for solving ILSs is developed. It is based on the straightforward idea of tracing labeled molecules through the metabolic network. The new approach allows to calculate the label distribution in isotopic tracer experiments in an analytical way that directly reflects the underlying network structure. The theory of path tracing is formally developed by introducing regular expressions for representing all possible paths through the labeling network. These expressions are generated by classical path tracing algorithms, e.g. by the Kleene algorithm. As a central theoretical result, a framework for proving the correctness of such path tracing algorithms in their application to ILSs is developed. Finally, by mapping path expressions to algebraic expressions, the solution of an ILS is computed. As an offspring of the developed theory, the relation between path tracing and former approaches for ILS solution is worked out and several consequences for the numerical solution and analysis of ILSs and - more general - compartmental systems used in pharmaco-kinetic modeling will be sketched. PMID:23507460

Wiechert, Wolfgang; Nöh, Katharina; Weitzel, Michael



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



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.

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



Amino acid selective labeling and unlabeling for protein resonance assignments.  


Structural characterization of proteins by NMR spectroscopy begins with the process of sequence specific resonance assignments in which the (1)H, (13)C and (15)N chemical shifts of all backbone and side-chain nuclei in the polypeptide are assigned. This process requires different isotope labeled forms of the protein together with specific experiments for establishing the sequential connectivity between the neighboring amino acid residues. In the case of spectral overlap, it is useful to identify spin systems corresponding to the different amino acid types selectively. With isotope labeling this can be achieved in two ways: (i) amino acid selective labeling or (ii) amino acid selective 'unlabeling'. This chapter describes both these methods with more emphasis on selective unlabeling describing the various practical aspects. The recent developments involving combinatorial selective labeling and unlabeling are also discussed. PMID:23076581

Jaipuria, Garima; Krishnarjuna, B; Mondal, Somnath; Dubey, Abhinav; Atreya, Hanudatta S



Evaluation of stable isotope tracing for ZnO nanomaterials--new constraints from high precision isotope analyses and modeling.  


This contribution evaluates two possible routes of stable isotope tracing for ZnO nanomaterials. For this we carried out the first high precision Zn isotope analyses of commercially available ZnO nanomaterials, to investigate whether such materials exhibit isotope fractionations that can be exploited for tracing purposes. These measurements revealed Zn isotopic compositions (of ?(66/64)Zn = +0.28 to -0.31‰ relative to JMC Lyon Zn) that are indistinguishable from "normal" natural and anthropogenic Zn in environmental samples. Stable isotope tracing therefore requires the application of purpose-made isotopically enriched ZnO nanoparticles. A detailed evaluation identified the most suitable and cost-effective labeling isotopes for different analytical requirements and techniques. It is shown that, using relatively inexpensive (68)Zn for labeling, ZnO nanoparticles can be reliably detected in natural samples with a Zn background of 100 ?g/g at concentrations as low as about 5 ng/g, if the isotopic tracing analyses are carried out by high precision mass spectrometry. Stable isotope tracing may also be able to differentiate between the uptake by organisms of particulate ZnO and Zn(2+) ions from the dissolution of nanoparticles. PMID:22394426

Larner, Fiona; Rehkämper, Mark



Tungsten isotope ratio determinations by negative thermal ionization mass spectrometry  

NASA Astrophysics Data System (ADS)

A precise determination of the isotopic abundances of tungsten with natural isotopic composition is presented. WO-3 ions are generated by negative thermal ionization (NTI) in a double-filament ion source. La2O3 is used as a chemical substance to reduce the electron work function of the rhenium filament material. An ionization efficiency of 1% is obtained for sample loadings of 100 ng. The isotopic abundances are measured with relative standard deviations of 0.2% for the least abundant 180W isotope and 0.02-0.004% for the other tungsten isotopes. These improved isotopic data are used to recalculate the atomic weight of tungsten as 183.8417 ± 0.0001. The new NTI technique is an ideal tool f