Time Course Of Labeling Research Articles

Characterization and optimization of fluorescein isothiocyanate labeling of humanized h2E2 anti-cocaine mAb

Fluorescein isothiocyanate (FITC) is widely used to fluorescently label reactive lysine residues on proteins, including antibodies. The rate and extent of labeling varies with reaction conditions, concentration of label, and the concentration and nature of the protein. Fluorescently labeled proteins are very useful, and one use for FITC labeled mAbs is development of assays to measure anti-mAb antibodies produced in vivo during treatment with antibody therapeutics. Our laboratory has developed a humanized anti-cocaine mAb (h2E2) intended for the treatment of cocaine use disorders. Thus, a well characterized FITC labeled h2E2 mAb is needed to quantitate possible anti-mAb antibodies. The time course of labeling and the relative incorporation of FITC into the heavy and light chains, as well as into the Fab and Fc portions of the mAb, was assessed. A novel use of differential scanning fluorimetry in the absence of any extrinsic fluorophore was developed and demonstrated to be capable of measuring antigen (cocaine) binding. In addition, the effect of increasing degrees of labeling by FITC on the thermodynamic parameters driving the binding of cocaine to the mAb was assessed via isothermal titration calorimetry (ITC). This binding technique, unlike others developed recently to measure cocaine binding, is not dependent on, or subject to interference by, the absorbance or fluorescence of the incorporated FITC label. The methods and results reported herein guide the optimization of FITC labeling needed for anti-mAb assays and other assays important for the development of therapeutic mAbs, which are some of the most specific and clinically useful drugs available.

GrandR: a comprehensive package for nucleotide conversion RNA-seq data analysis

Metabolic labeling of RNA is a powerful technique for studying the temporal dynamics of gene expression. Nucleotide conversion approaches greatly facilitate the generation of data but introduce challenges for their analysis. Here we present grandR, a comprehensive package for quality control, differential gene expression analysis, kinetic modeling, and visualization of such data. We compare several existing methods for inference of RNA synthesis rates and half-lives using progressive labeling time courses. We demonstrate the need for recalibration of effective labeling times and introduce a Bayesian approach to study the temporal dynamics of RNA using snapshot experiments.

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry.

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

Reimport of carbon from cytosolic and vacuolar sugar pools into the Calvin–Benson cycle explains photosynthesis labeling anomalies

SignificancePhotosynthesis metabolites are quickly labeled when 13CO2 is fed to leaves, but the time course of labeling reveals additional contributing processes involved in the metabolic dynamics of photosynthesis. The existence of three such processes is demonstrated, and a metabolic flux model is developed to explore and characterize them. The model is consistent with a slow return of carbon from cytosolic and vacuolar sugars into the Calvin-Benson cycle through the oxidative pentose phosphate pathway. Our results provide insight into how carbon assimilation is integrated into the metabolic network of photosynthetic cells with implications for global carbon fluxes.

The metabolic origins of non-photorespiratory CO2 release during photosynthesis: a metabolic flux analysis.

Respiration in the light (RL) releases CO2 in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since RL lowers net carbon fixation, understanding RL could help improve plant carbon-use efficiency and models of crop photosynthesis. Although RL was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to RL, we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during 13CO2 labeling time course, gas exchange, and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO2 sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N2; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that RL releases 5.2 μmol CO2 g-1 FW h-1 of CO2, which is relatively consistent with a value of 9.3 μmol CO2 g-1 FW h-1 measured by CO2 gas exchange. The results indicated that ≤10% of RL results from TCA cycle reactions, which are widely considered to dominate RL. Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >93% of CO2 released by RL.

Large fluxes of fatty acids from membranes to triacylglycerol and back during N-deprivation and recovery in Chlamydomonas.

Microalgae accumulate triacylglycerol (TAG) during nutrient deprivation and break it down after nutrient resupply, and these processes involve dramatic shifts in cellular carbon allocation. Due to the importance of algae in the global carbon cycle, and the potential of algal lipids as feedstock for chemical and fuel production, these processes are of both ecophysiological and biotechnological importance. However, the metabolism of TAG is not well understood, particularly the contributions of fatty acids (FAs) from different membrane lipids to TAG accumulation and the fate of TAG FAs during degradation. Here, we used isotopic labeling time course experiments on Chlamydomonas reinhardtii to track FA synthesis and transfer between lipid pools during nitrogen (N)-deprivation and resupply. When cells were labeled before N-deprivation, total levels of label in cellular FAs were unchanged during subsequent N-deprivation and later resupply, despite large fluxes into and out of TAG and membrane lipid pools. Detailed analyses of FA levels and labeling revealed that about one-third of acyl chains accumulating in TAG during N-deprivation derive from preexisting membrane lipids, and in total, at least 45% of TAG FAs passed through membrane lipids at one point. Notably, most acyl chains in membrane lipids during recovery after N-resupply come from TAG. Fluxes of polyunsaturated FAs from plastidic membranes into TAG during N-deprivation were particularly noteworthy. These findings demonstrate a high degree of integration of TAG and membrane lipid metabolism and highlight a role for TAG in storage and supply of membrane lipid components.

Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

Dynamic 1 H-MRS for detection of 13 C-labeled glucose metabolism in the human brain at 3T.

In 2004, Boumezbeur et al proposed a simple yet powerful approach to detect the metabolism of 13 C-enriched substrates in the brain. Their approach consisted of dynamic 1 H-MRS, without a 13 C radiofrequency (RF) channel, and its successful application was demonstrated in monkeys. Since then, this promising method has yet to be applied rigorously in humans. In this study, we revisit the use of dynamic 1 H-MRS to measure the metabolism of 13 C-enriched substrates and demonstrate its application in the human brain. In healthy participants, 1 H-MRS data were acquired dynamically before and following a bolus infusion of [1-13 C] glucose. Data were acquired on a 3T clinical MRI scanner using a short-TE SPECIAL sequence, with regions of interest in both anterior and posterior cingulate cortex. Using simulated basis spectra to model signal changes in both 12 C-bonded and 13 C-coupled resonances, the acquired spectra were fit in LCModel to obtain labeling time courses for glutmate and glutamine at both C4 and C3 positions. Presence of the 13 C label was clearly detectable, owing to the pronounced effect of heteronuclear (13 C-1 H) scalar coupling on the observed 1 H spectra. A decrease in signal from 12 C-bonded protons and an increase in signal from 13 C-coupled protons were observed. The fractional enrichment of Glu-C4, (Glu+Gln)-C4, and (Glu+Gln)-C3 at 30 minutes following infusion of [1-13 C] glucose was similar in both regions: 11% to 13%, 9% to 12% and 3% to 5%, respectively. These preliminary results confirm the feasibility of the use of dynamic 1 H-MRS to monitor 13 C labeling in the human brain, without a 13 C RF channel.

Use of MALDI-MS with solid-state hydrogen deuterium exchange for semi-automated assessment of peptide and protein physical stability in lyophilized solids

Biological therapeutics are established as major contributors to the pharmaceutical pipeline. Many of these biological drugs are lyophilized to preserve their conformation and reduce decomposition during storage and shipping. Therefore, understanding and controlling the effects of lyophilization on protein higher order structure is critical for commercialization of biologics. Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a well-established technique for studying protein higher order structure. Previous publications have demonstrated a solid state HDX (ssHDX) method for labeling formulated, lyophilized proteins to assess their physical stability during, but this process still suffered from low throughput and undesired back exchange. Recently, our group described a method combining HDX-MS with MALDI to greatly reduce the time of analysis and nearly eliminate H/D back-exchange, but that method was not suited for interrogating solid samples. This work integrates the two techniques to assess and predict the stability of peptides and proteins following mixing and lyophilization with various excipient formulations. Sample mixing and handling were performed through the use of a bench-top robotics and programmed data MALDI-MS acquisition allowed for monitoring deuterium incorporation for dried peptides and protein samples following continuous labeling with D2O vapor. Effects of excipients upon peptide stability were also tracked and compared to a control for a three day labeling time course. This workflow is automated and free from back-exchange. As demonstrated by deuterium retention of bradykinin, these features serve to reduce experimental error normally associated with conventional deuterium exchange experiments. The proposed union of MALDI-MS and ssHDX can be applied to study higher order structure of proteins and peptides and the effects of added excipients in an environment that closely resembles the storage and shipping conditions of biopharmaceuticals and may be beneficial in giving insights studying protein structural dynamics in solids.

The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows during Nutrient Deprivation in Chlamydomonas reinhardtii.

Because of the potential importance of algae for green biotechnology, considerable effort has been invested in understanding their responses to nitrogen deprivation. The most frequently invoked reasons proposed for the accumulation of high cellular levels of triacylglycerol (TAG) and starch are variants of what may be termed the "overflow hypothesis." According to this, growth inhibition results in the rate of photosynthetic energy and/or carbon input exceeding cellular needs; the excess input is directed into the accumulation of TAG and/or starch to prevent damage. This study was aimed at providing a quantitative dataset and analysis of the main energy and carbon flows before and during nitrogen deprivation in a model system to assess alternative explanations. Cellular growth, biomass, starch, and lipid levels as well as several measures of photosynthetic function were recorded for cells of Chlamydomonas reinhardtii cultured under nine different autotrophic, mixotrophic, and heterotrophic conditions during nutrient-replete growth and for the first 4 d of nitrogen deprivation. The results of a (13)C labeling time course indicated that in mixotrophic culture, starch is predominantly made from CO2 and fatty acid synthesis is largely supplied by exogenous acetate, with considerable turnover of membrane lipids, so that total lipid rather than TAG is the appropriate measure of product accumulation. Heterotrophic cultures accumulated TAG and starch during N deprivation, showing that these are not dependent on photosynthesis. We conclude that the overflow hypothesis is insufficient and suggest that storage may be a more universally important reason for carbon compound accumulation during nutrient deprivation.

Refined Analysis of Brain Energy Metabolism Using In Vivo Dynamic Enrichment of 13C Multiplets.

Carbon-13 nuclear magnetic resonance spectroscopy in combination with the infusion of 13C-labeled precursors is a unique approach to study in vivo brain energy metabolism. Incorporating the maximum information available from in vivo localized 13C spectra is of importance to get broader knowledge on cerebral metabolic pathways. Metabolic rates can be quantitatively determined from the rate of 13C incorporation into amino acid neurotransmitters such as glutamate and glutamine using suitable mathematical models. The time course of multiplets arising from 13C-13C coupling between adjacent carbon atoms was expected to provide additional information for metabolic modeling leading to potential improvements in the estimation of metabolic parameters.The aim of the present study was to extend two-compartment neuronal/glial modeling to include dynamics of 13C isotopomers available from fine structure multiplets in 13C spectra of glutamate and glutamine measured in vivo in rats brain at 14.1 T, termed bonded cumomer approach. Incorporating the labeling time courses of 13C multiplets of glutamate and glutamine resulted in elevated precision of the estimated fluxes in rat brain as well as reduced correlations between them.

Dynamic metabolic flux analysis of plant cell wall synthesis

The regulation of plant cell wall synthesis pathways remains poorly understood. This has become a bottleneck in designing bioenergy crops. The goal of this study was to analyze the regulation of plant cell wall precursor metabolism using metabolic flux analysis based on dynamic labeling experiments. Arabidopsis T87 cells were cultured heterotrophically with 13C labeled sucrose. The time course of 13C labeling patterns in cell wall precursors and related sugar phosphates was monitored using liquid chromatography tandem mass spectrometry until steady state labeling was reached. A kinetic model based on mass action reaction mechanisms was developed to simulate the carbon flow in the cell wall synthesis network. The kinetic parameters of the model were determined by fitting the model to the labeling time course data, cell wall composition, and synthesis rates. A metabolic control analysis was performed to predict metabolic regulations that may improve plant biomass composition for biofuel production. Our results describe the routes and rates of carbon flow from sucrose to cell wall precursors. We found that sucrose invertase is responsible for the entry of sucrose into metabolism and UDP-glucose-4-epimerase plays a dominant role in UDP-Gal synthesis in heterotrophic Aradidopsis cells under aerobic conditions. We also predicted reactions that exert strong regulatory influence over carbon flow to cell wall synthesis and its composition.

A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and 13C-labeled glucose

Metabolic models have been used to elucidate important aspects of brain metabolism in recent years. This work applies for the first time the concept of isotopic transient 13C metabolic flux analysis (MFA) to estimate intracellular fluxes in primary cultures of astrocytes. This methodology comprehensively explores the information provided by 13C labeling time-courses of intracellular metabolites after administration of a 13C-labeled substrate. Cells were incubated with medium containing [1-13C]glucose for 24 h and samples of cell supernatant and extracts collected at different time points were then analyzed by mass spectrometry and/or high performance liquid chromatography. Metabolic fluxes were estimated by fitting a carbon labeling network model to isotopomer profiles experimentally determined. Both the fast isotopic equilibrium of glycolytic metabolite pools and the slow labeling dynamics of TCA cycle intermediates are described well by the model. The large pools of glutamate and aspartate which are linked to the TCA cycle via reversible aminotransferase reactions are likely to be responsible for the observed delay in equilibration of TCA cycle intermediates. Furthermore, it was estimated that 11% of the glucose taken up by astrocytes was diverted to the pentose phosphate pathway. In addition, considerable fluxes through pyruvate carboxylase [PC; PC/pyruvate dehydrogenase (PDH) ratio = 0.5], malic enzyme (5% of the total pyruvate production), and catabolism of branched-chained amino acids (contributing with ∼40% to total acetyl-CoA produced) confirmed the significance of these pathways to astrocytic metabolism. Consistent with the need of maintaining cytosolic redox potential, the fluxes through the malate–aspartate shuttle and the PDH pathway were comparable. Finally, the estimated glutamate/α-ketoglutarate exchange rate (∼0.7 μmol mg prot−1 h−1) was similar to the TCA cycle flux. In conclusion, this work demonstrates the potential of isotopic transient MFA for a comprehensive analysis of energy metabolism.

The Use of PRV-Bartha to Define Premotor Inputs to Lumbar Motoneurons in the Neonatal Spinal Cord of the Mouse

BackgroundThe neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.Methodology/Principal FindingsHere we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24–32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32–40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8–12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.Conclusions/SignificanceThe study establishes the time window for virally - labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks.

Prevention of Amino Acid Conversion in SILAC Experiments with Embryonic Stem Cells

Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotope-coded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter L-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human.

In vivo 13C NMR spectroscopy and metabolic modeling in the brain: a practical perspective

In vivo 13C NMR spectroscopy has the unique capability to measure metabolic fluxes noninvasively in the brain. Quantitative measurements of metabolic fluxes require analysis of the 13C labeling time courses obtained experimentally with a metabolic model. The present work reviews the ingredients necessary for a dynamic metabolic modeling study, with particular emphasis on practical issues.

Regulation of parietal cell migration by gastrin in the mouse.

Recent studies suggest that gastrin regulates parietal cell maturation. We asked whether it also regulates parietal cell life span and migration along the gland. Dividing cells were labeled with 5'-bromo-2'-deoxyuridine (BrdU), and parietal cells were identified by staining with Dolichos biflorus lectin. Cells positive for D. biflorus lectin and BrdU were reliably identified 10-30 days after BrdU injection in mice in which the gastrin gene had been deleted by homologous recombination (Gas-KO) and wild-type (C57BL/6) mice. The time course of labeling was similar in the two groups. The distribution of BrdU-labeled parietal cells in wild-type mice was consistent with migration to the base of the gland, but in Gas-KO mice, a higher proportion of BrdU-labeled cells was found more superficially 20 and 30 days after BrdU injection. Conversely, in transgenic mice overexpressing gastrin, BrdU-labeled parietal cells accounted for a higher proportion of the labeled pool in the base of the gland 10 days after BrdU injection. Gastrin, therefore, stimulates movement of parietal cells along the gland axis but does not influence their life span.

Measurement of the tricarboxylic acid cycle rate in human grey and white matter in vivo by 1H-[13C] magnetic resonance spectroscopy at 4.1T.

13C isotopic labeling data were obtained by 1H-observed/13C-edited magnetic resonance spectroscopy in the human brain in vivo and analyzed using a mathematical model to determine metabolic rates in human grey matter and white matter. 22.5-cc and 56-cc voxels were examined for grey matter and white matter, respectively. When partial volume effects were ignored, the measured tricarboxylic acid cycle rate was 0.72+/-0.22 (mean +/- SD) and 0.29+/-0.09 micromol min(-1) g(-1) (mean +/- SD) in voxels of approximately 70% grey and approximately 70% white matter, respectively. After correction for partial volume effects using a model with two tissue compartments, the tricarboxylic acid cycle rate in pure grey matter was higher (0.80+/-0.10 mol min(-1) g(-1); mean +/- SD) and in white matter was significantly lower (0.17+/-0.01 micromol min(-1) g(-1); mean +/- SD). In 1H-observed/13C-edited magnetic resonance spectroscopy labeling studies, the larger concentrations of labeled metabolites and faster metabolic rates in grey matter biased the measurements heavily toward grey matter, with labeling time courses in 70% grey matter appearing nearly identical to labeling in pure grey matter.

Evidence of the Photosynthetic Origin of Monoterpenes Emitted by Quercus ilex L. Leaves by 13C Labeling.

The carbon of the four main monoterpenes emitted by Quercus ilex L. leaves was completely labeled with 13C after a 20-min feeding with 99% 13CO2. This labeling time course is comparable with the labeling time course of isoprene, the terpenoid emitted by other Quercus species and synthesized in leaf chloroplasts. It is also comparable with that of phosphoglyceric acid. Our experiment therefore provides evidence that monoterpenes emitted by Q. ilex are formed from photosynthesis intermediates and may share the same synthetic pathway with isoprene. By analyzing the rate and the distribution of labeling in the different fragments, we looked for evidence of differential carbon labeling in the [alpha]-pinene emitted. However, the labeling pattern was quite uniform in the different fragments, suggesting that the carbon skeleton of the emitted monoterpenes comes from a unique carbon source.