Literatura científica selecionada sobre o tema "Continuous isotope labeling"

Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This “metabolic flux probe” (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.

ABSTRACT Porphyromonas gingivalis is an anaerobic, asaccharolytic, gram-negative bacterium that has essential requirements for both iron and protoporphyrin IX, which it preferentially obtains as heme. A combination of large-scale quantitative proteomic analysis using stable isotope labeling strategies and mass spectrometry, together with transcriptomic analysis using custom-made DNA microarrays, was used to identify changes in P. gingivalis W50 protein and transcript abundances on changing from heme-excess to heme-limited continuous culture. This approach identified 160 genes and 70 proteins that were differentially regulated by heme availability, with broad agreement between the transcriptomic and proteomic data. A change in abundance of the enzymes of the aspartate and glutamate catabolic pathways was observed with heme limitation, which was reflected in organic acid end product levels of the culture fluid. These results demonstrate a shift from an energy-efficient anaerobic respiration to a less efficient process upon heme limitation. Heme limitation also resulted in an increase in abundance of a protein, PG1374, which we have demonstrated, by insertional inactivation, to have a role in epithelial cell invasion. The greater abundance of a number of transcripts/proteins linked to invasion of host cells, the oxidative stress response, iron/heme transport, and virulence of the bacterium indicates that there is a broad response of P. gingivalis to heme availability.

Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by SAM for decades, but the importance of this regulatory control to one-carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one-carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transsulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. 13C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CH3THF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one-carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one-carbon metabolism with various pathways both in yeasts and in humans.

A new two-column 225Ac/213Bi generator was developed specifically for using 225Ac containing an impurity of long lived 227Ac. The parent 225Ac was retained on the first Actinide Resin column, while 213Bi was accumulated on the second column filled with AG MP-50 resin via continuous elution and decay of intermediate 221Fr. The 213Bi accumulation was realized in circulation mode which allowed a compact generator design. It was demonstrated that 213Bi could be quickly and effectively extracted from AG MP-50 in form of complexes with various chelating agents including DTPA and DOTA. The performance of the generator presented and a conventional single-column generator on the base of AG MP-50 was tested and both generators were loaded with 225Ac containing 227Ac impurity. The 213Bi generation efficiencies were comparable and greater than 70%, whereas the developed generator provided a deeper degree of purification of 213Bi from Ac isotopes and decay products of 227Ac.

Although theoretically all glycoprotein sugars can be derived from glucose, it may be hypothesized that specific dietary sugars could be preferential substrates for glycoprotein synthesis. To test this hypothesis, groups of rats received either continuously (continuous-labelling experiment) or for a single nutritional period (pulse-labelling experiment) a 13C-rich diet containing either maize starch or artificially labelled [13C]glucose. Some groups of rats were also provided during a single nutritional period with low amounts (20–200 mg/animal) of low-13C dietary sugars (mannose, galactose, fucose or fructose). If specific dietary sugars were preferentially incorporated into glycoproteins instead nf glucose-derived labelled sugars, a decrease would be expected in the intestinal or serum glycoprotein-sugar 13C enrichment monitored by gas chromatography-isotope-ratio mass spectrometry (GC-IRMS). Contrary to this hypothesis the results showed no significant decrease with any of the specific dietary sugars. Furthermore, with dietary low-13C mannose or galactose, a significant increase in 13C enrichment of glycoprotein-sugars was observed compared with some other nutritional groups. Moreover, in the pulse-labelling experiment, dietary mannose and galactose induced similar patterns of 13C enrichment in intestinal and serum glycoprotein-sugars. Therefore, although specific dietary sugars do not appear to be preferential substrates for glycosylation under conditions and doses relevant to current concepts of nutrition, regulatory roles of some specific dietary sugars in relation to glycoprotein-sugar metabolism might be hypothesized. These findings could lead to similar studies using stable-isotope methodology in man which could have practical consequences, especially in parenteral nutrition where glucose is the only sugar provided to the metabolism.

The accurate quantitation of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics. Mass spectrometry-based approaches have forged significant in-roads allowing accurate and sensitive quantitation and the ability to multiplex vastly complex samples through the application of robust bioinformatic tools. These relative and absolute quantitative measures using label-free, tags, or stable isotope labelling have their own strengths and limitations. The continuous development of these methods is vital for increasing reproducibility in the rapidly expanding application of quantitative proteomics in biomarker discovery and validation. This paper provides a critical overview of the primary mass spectrometry-based quantitative approaches and the current status of quantitative proteomics in biomedical research.

Muscle protein synthesis (MPS) rate is determined conventionally by obtaining two or more tissue biopsies during a primed, continuous infusion of a stable isotopically labeled amino acid. The purpose of the present study was to test whether tracer priming given as a flooding dose, thereby securing an instantaneous labeling of the tissue pools of free tracee amino acids, followed by a continuous infusion of the same tracer to maintain tracer isotopic steady state, could be used to determine the MPS rate over a prolonged period of time by obtaining only a single tissue biopsy. We showed that the tracer from the flood prime appeared immediately in the muscle free pool of amino acids and that this abundance could be kept constant by a subsequent continuous infusion of the tracer. When using phenylalanine as tracer, the flood-primed, continuous infusion protocol does not stimulate the MPS rate per se. In conclusion, the flood-primed, continuous infusion protocol using phenylalanine as tracer can validly be used to measure the protein synthesis rate in human in vivo experiments by obtaining only a single tissue biopsy after a prolonged infusion period.

Abstract. Despite the importance of carbon (C) pools and CO2 fluxes in terrestrial ecosystems and especially in soils, as well as many attempts to assign fluxes to specific pools, this challenge remains unsolved. Interestingly, scientists investigating pools are not closely linked with scientists studying fluxes. This mini-review therefore focused on experimental approaches enabling soil C pools to be linked with CO2 flux from the soil. The background, advantages and shortcomings of uncoupled approaches (measuring only pools or fluxes) and of coupled approaches (measuring both pools and fluxes) were evaluated and their prerequisites – steady state of pools and isotopic steady state – described. The uncoupled approaches include: (i) monitoring the decrease of C pools in long-term fallow bare soil lacking C input over decades, (ii) analyzing components of CO2 efflux dynamics by incubating soil without new C input over months or a few years, and (iii) analyzing turnover rates of C pools based on their 13C and 14C isotopic signature. The uncoupled approaches are applicable for non steady state conditions only and have limited explanatory power. The more advantageous coupled approaches partition simultaneously pools and fluxes and are based on one of three types of changes in the isotopic signature of input C compared to soil C: (i) abrupt permanent, (ii) gradual permanent, and (iii) abrupt temporary impacts. I show how the maximal sensitivity of the approaches depends on the differences in the isotopic signature of pools with fast and slow turnover rates. The promising coupled approaches include: (a) &delta13C of C pools and CO2 efflux from soil after C3/C4 vegetation changes or in FACE experiments (both corresponding to continuous labeling), (b) addition of 13C or 14C labeled organics (corresponding to pulse labeling), and (c) bomb-14C. I show that physical separation of soil C pools is not a~prerequisite to estimate pool size or to link pools with fluxes. The future challenges include combining two or more promising approaches to elucidate more than two C sources for CO2 fluxes, and linking scientific communities investigating the pools with those investigating the fluxes.

The simultaneous photocatalytic H2 evolution with environmental remediation over semiconducting metal oxides is a fascinating process for sustainable fuel production. However, most of the previously reported photocatalytic reforming showed nonstoichiometric amounts of the evolved H2 when organic substrates were used. To explain the reasons for this phenomenon, a careful analysis of the products and intermediates in gas and aqueous phases upon the photocatalytic hydrogen evolution from oxalic acid using Pt/TiO2 was performed. A quadrupole mass spectrometer (QMS) was used for the continuous flow monitoring of the evolved gases, while high performance ion chromatography (HPIC), isotopic labeling, and electron paramagnetic resonance (EPR) were employed to understand the reactions in the solution. The entire consumption of oxalic acid led to a ~30% lower H2 amount than theoretically expected. Due to the contribution of the photo-Kolbe reaction mechanism, a tiny amount of formic acid was produced then disappeared shortly after the complete consumption of oxalic acid. Nevertheless, a much lower concentration of formic acid was generated compared to the nonstoichiometric difference between the formed H2 and the consumed oxalic acid. Isotopic labeling measurements showed that the evolved H2, HD, and/or D2 matched those of the solvent; however, using D2O decreased the reaction rate. Interestingly, the presence of KI as an additional hole scavenger with oxalic acid had a considerable impact on the reaction mechanism, and thus the hydrogen yield, as indicated by the QMS and the EPR measurements. The added KI promoted H2 evolution to reach the theoretically predictable amount and inhibited the formation of intermediates without affecting the oxalic acid degradation rate. The proposed mechanism, by which KI boosts the photocatalytic performance, is of great importance in enhancing the overall energy efficiency for hydrogen production via photocatalytic organic reforming.

L'augmentation des émissions anthropiques de CO2 dans l'atmosphère accélère le changement climatique. Les sols contiennent trois fois plus de carbone que l'atmosphère et constituent donc un réservoir d'importance cruciale pour la régulation du climat. Il existe actuellement une réflexion pour stocker le carbone dans les couches profondes du sol, notamment via la rhizodéposition des plantes. Nous avons donc mené une expérience au CEREEP-Ecotron Ile-de-France pour quantifier les apports, et la persistance, du carbone rhizodéposé par les plantes à l'aide d'un marquage continu au 13C-CO2. Pour ce faire, deux variétés de blé aux systèmes racinaires contrastés ont été plantés dans des mésocosmes et cultivés pendant une saison de croissance complète et sous atmosphère enrichie en 13C. Nos objectifs étaient de quantifier le flux de carbone de l'atmosphère vers le sol et de mesurer sa persistance à court terme. Nos résultats suggèrent que la variété ancienne Plantahof rhizodépose une quantité plus élevée de carbone par rapport à la variété récente Nara notamment en profondeur. Cependant, le carbone apporté au sol par ces deux variétés a conduit à des pertes par minéralisation et des priming effects similaires. Ainsi, le bilan total du carbone était plus affecté par la profondeur du sol que les variétés utilisées dans l'étude. Par ailleurs, j'ai étudié, à partir d'une analyse bibliographique, la distribution selon la profondeur des activités enzymatiques hydrolases et oxydoréductases impliquées dans les cycles du carbone, de l'azote et du phosphore en fonction de la profondeur du sol. Les résultats de cette analyse ont montré que les profils d'activité dépendaient très fortement de la façon dont ces activités étaient exprimées, avec des activités qui diminuent avec la profondeur lorsqu'exprimées par masse de sol alors qu'elles sont plutôt stables, voire augmentent, lorsque exprimé par rapport à la biomasse microbienne. Pris dans leur ensemble, ces résultats montrent que la prise en compte du fonctionnement sur l'intégralité de la colonne de sol est indispensable pour comprendre la dynamique du carbone dans les écosystèmes terrestres
Increasing anthropogenic emissions of CO2 to the atmosphere are accelerating climate change. These emissions could be partially compensated by carbon fixation in the oceans, vegetation and soils. In particular, soils contain three times more carbon than the atmosphere, and therefore play a crucial role in climate regulation. It has been suggested that storing carbon in the deep layers of the soil, via rhizodeposition of plants, may be a useful avenue to pursue in order to mitigate climate change. We therefore conducted an experiment at CEREEP-Ecotron Ile-de-France to quantify the input and persistence of rhizodeposited carbon by plants using a continuous 13C-CO2 label. Two wheat varieties with contrasting root systems were planted in mesocosms and grown for a full growing season in a 13C-enriched atmosphere. Our objectives were to quantify the rooting-dependent flux of carbon from the atmosphere to the soil by isotopic tracing with 13C, and to measure its short-term persistence. The results showed that the old variety Plantahof rhizodeposited a larger amount of carbon than the more recent variety Nara, especially at depth. However, the carbon supplied to the soil by these two varieties led to similar amounts of organic C mineralization and priming effects. Thus, the total carbon balance was more related to the effect of soil depth than to the varieties used in the study. Furthermore, I carried out a meta-analysis of the distribution of enzymatic activities as a function of soil depth for hydrolases and oxidoreductases involved in the carbon, nitrogen and phosphorus cycle. The results of this analysis showed that the activity profiles depended very strongly on the way these activities were expressed, with activities mostly decreasing when expressed per soil mass, but remaining rather stable or even increasing with depth when expressed per unit microbial biomass. Taken together, these results show that considering the functioning of the entire soil column is essential to understand the dynamics of carbon in terrestrial ecosystems

Abstract “Heavy Isotope Enrichments” reviews concepts for developing and interpreting heavy isotope labeling studies in ecology. The chapter compares continuous- and pulse-labeling approaches for the 13C labeling of plant material. Methods for the application of isotope-labeled substrates and their use in stable isotope probing of biological markers are presented. Calculations of the heavy isotope excess and the quantification of the recovery of heavy isotope additions in isotope tracing studies are provided. Principles and equations for the application of the isotope dilution method are illustrated, and its limitations are discussed. Examples of applications for carbon, nitrogen, and water stable isotopes are presented.