Steady-state Labeling Research Articles

Biochar Amendment Stimulates Utilization of Plant-Derived Carbon by Soil Bacteria in an Intercropping System.

Plant-derived carbon (C) is considered fundamental to understand the interaction between rhizosphere microbes and plants in terrestrial ecosystems. Biochar soil amendment may enhance plant performance via changing soil properties or microbial diversity in the rhizosphere. However, our knowledge of how plant-microbiome associations respond to biochar amendment remains rather limited. Herein, 13CO2 steady-state labeling combined with DNA stable-isotope probing was used to characterize soil bacterial communities in the rhizosphere contributing to the utilization of plant-derived C. The diversity of bacteria active in the utilization of root exudates was determined after biochar amendment in a legume-based intercropping system (Vicia faba L., with Zea mays L.). The results showed the biochar application not only changed the bacterial community structure and diversity in the rhizosphere, but also altered bacterial members actively assimilating plant-derived C. There were more labeled species in the biochar-amended soils than the control soils. Compared with the control, the biochar amendment increased the relative abundances of Firmicutes and Bacteroidetes members (i.e., Bacillus, Clostridium, Sporomusa, Desulfosporosinus, and Alicyclobacillus) while decreasing the abundances of Proteobacteria members (e.g., Methylobacterium and Sphingomonas) utilizing plant-derived C. In contrast, slow-growing species of the phyla Acidobacteria, Planctomycetes, and Gemmatimonadetes were barely labeled. The bacteria found stimulated by the biochar amendment are known for their ability to fix nitrogen, solubilize phosphorus, or reduce iron and sulfur, which may potentially contribute to the “biochar effect” in the rhizosphere. This study is the first to provide empirical evidence that biochar amendment can alter the soil bacterial community assimilating plant-derived C; this may have consequences for nutrient cycling and improving plant performance in intercropping systems.

Stable Isotope Tracers for Metabolic Pathway Analysis.

Stable isotope tracing allows a metabolic substrate to be followed through downstream biochemical reactions, thereby providing unparalleled insights into the metabolic wiring of cells. This approach stops short of modeling absolute fluxes but is relatively straightforward and has become increasingly accessible due to the widespread adoption of high-resolution mass spectrometers. Analysis of both dynamic and steady-state labeling patterns in downstream metabolites provides valuable qualitative information as to their origin and relative rates of production. Stable isotope tracing is, therefore, a powerful way to understand the impact of genetic alterations and defined perturbations on metabolism. In this chapter, we describe a liquid chromatography-mass spectrometry (LC-MS) protocol for stable isotope tracing using 13C-L-arginine in a macrophage cell line. A similar approach can be used to follow other stable isotope tracers, and notes are provided with advice on how this protocol can be generalized for use in other settings.

Effects of Light Quality and Intensity on Diurnal Patterns and Rates of Photo-Assimilate Translocation and Transpiration in Tomato Leaves.

Translocation of assimilates is a fundamental process involving carbon and water balance affecting source/sink relationships. Diurnal patterns of CO2 exchange, translocation (carbon export), and transpiration of an intact tomato source leaf were determined during 14CO2 steady-state labeling under different wavelengths at three pre-set photosynthetic rates. Daily patterns showed that photosynthesis and export were supported by all wavelengths of light tested including orange and green. Export in the light, under all wavelengths was always higher than that at night. Export in the light varied from 65–83% of the total daily carbon fixed, depending on light intensity. Photosynthesis and export were highly correlated under all wavelengths (r = 0.90–0.96). Export as a percentage of photosynthesis (relative export) decreased as photosynthesis increased by increasing light intensity under all wavelengths. These data indicate an upper limit for export under all spectral conditions. Interestingly, only at the medium photosynthetic rate, relative export under the blue and the orange light-emitting diodes (LEDs) were higher than under white and red-white LEDs. Stomatal conductance, transpiration rates, and water-use-efficiency showed similar daily patterns under all wavelengths. Illuminating tomato leaves with different spectral quality resulted in similar carbon export rates, but stomatal conductance and transpiration rates varied due to wavelength specific control of stomatal function. Thus, we caution that the link between transpiration and C-export may be more complex than previously thought. In summary, these data indicate that orange and green LEDs, not simply the traditionally used red and blue LEDs, should be considered and tested when designing lighting systems for optimizing source leaf strength during plant production in controlled environment systems. In addition, knowledge related to the interplay between water and C-movement within a plant and how they are affected by environmental stimuli, is needed to develop a better understanding of source/sink relationships.

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids.

Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2 Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [(13)C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids. Biological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N2 However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-β-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules.

Abstract A14: 13C metabolomic and fluxomic study of human B-cell lymphoma metabolic network: Quantifying glutaminolysis and reductive carboxylation

Abstract Intracellular 13C LC-MS and MRS isotopomer data coupled with metabolic modeling allows quantitative determination of metabolic fluxes and other metabolic parameters in vivo/in situ (1). Utilizing these methods, we have determined how an mTOR inhibitor, rapamycin, alters glycolysis, the TCA cycle, the pentose phosphate pathway (PPP) and glutamine metabolism in B-cell lymphoma cell lines. This analysis has revealed previously unknown pharmacological activities for rapamycin. DLCL2 (germinal-center type of diffuse large B-cell lymphoma) and Ramos (Burkitt’s lymphoma resembling activated ABC type of B-cells) cells were incubated in glucose and glutamine free DMEM containing 10% fetal bovine serum, 5 mM unlabeled or 13C-labelled glucose, 2 mM unlabeled or [U-13C5, 15N2] glutamine and rapamycin for 6 h. The steady-state labeling patterns of TCA cycle metabolites (LC-MS) and temporal profiles for labeled lactate and glutamate (NMR) were monitored. Data were analyzed with compartmentalized metabolic network models that included glucose, lactate and glutamine transport, glycolysis, TCA cycle, PPP, α-ketoglutarate-glutamate and oxaloacetate-aspartate exchange, pyruvate carboxylase activity, anaplerosis at the succinyl-CoA level, pyruvate recycling through malic enzyme, lactate dehydrogenase activity, glutaminolysis, reductive carboxylation and de novo fatty acids synthesis. Isotope balance equations were derived for every network metabolite. The resulted sets of differential equations were solved to determine flux through various metabolic pathways by isotopomer/cumomer analysis (2). Statistics for the obtained parameters/fluxes was calculated by Monte Carlo simulations including experimental noise levels. We analyzed changes in expression of ~10,000 metabolic genes in DLCL2 and Ramos cells induced by rapamycin (by RNA-seq). Anticipated related alterations indicate that mTOR inhibition diminishes glycolysis, TCA cycle activity, de novo fatty acid biosynthesis, pyruvate cycling and PPP. Glutaminolisys was also affected due to gene PPAT expression (responsible for enzymatic glutamate production from glutamine). Metabolic Flux Analysis revealed that in DLCL2 rapamycin produced dramatic metabolic changes in reductive carboxylation (41% decrease), glutaminolysis (95% decrease), mitochondrial pyruvate carrier (50% increase) and pyruvate carboxylase (520% increase) activities. Further study of these effects is required, yet these data strongly suggest that multiple pathways are affected and, potentially, these metabolic signatures may serve as key biomarkers of effective mTOR inhibition. Our genomic and proteomic analysis indicates that the expression level of a large number of enzymes in human lymphoma cells has been modified by rapamycin-mediated mTOR inhibition. These affected enzymes are involved in eight metabolic pathways: glycolysis, pyruvate cycling/metabolism, TCA cycle, glutaminolysis, fatty acid metabolism, cholesterol metabolism, phospholipid metabolism and the pentose shunt. Information available from 13C multiplets in 13C spectra and MID of numerous metabolites significantly increases precision of calculated metabolic fluxes and allows estimation of contributions of different pathways to particular metabolite labeling patterns. The new modeling approaches developed here provide more effecient methods for investigation of detailed mechanisms of cancer metabolism and therefore enable effective identification of targets for combinatorial therapy. Dynamic high-resolution MR spectra and LC-MS mass-isotopomers are very sensitive to changing/adding biochemical pathways and flux values, and metabolic modeling allows one to check precisely the feasibility of assumed general bionetworks and alterations of particular metabolic pathways.

Optimization of non-contrast-enhanced MR angiography of the renal artery with three-dimensional balanced steady-state free-precession and time-spatial labeling inversion pulse (time-SLIP) at 3T MRI, in relation to age and blood velocity.

To determine the optimal inversion time (TI) value of three-dimensional (3D) balanced steady-state free-precession time-spatial labeling inversion pulse (time-SLIP) technique for visualization of the renal artery at 3T MRI, and to assess whether the optimal TI is affected by the subject's age and blood velocity. Forty-two healthy volunteers (range 20-67 years) were enrolled in the study and subjected to non-contrast-enhanced renal MR angiography. Five different TI values (1200, 1400, 1600, 1800, and 2000 ms) were selected for evaluation. For quantitative evaluation, the relative signal intensity (SI) of the main renal artery was compared with that of the renal medulla (Vessel-to-Kidney ratio; VKR). Blood velocity of the abdominal aorta was measured using 2D phase contrast technique. For qualitative evaluation, two radiologists scored the depiction of the renal pelvis and the quality of visualization of the renal artery. VKR is the highest at TI = 1600 ms. A strong negative correlation between age and blood velocity was demonstrated. Regarding the qualitative evaluation, the overall image scores of renal arteries were the highest at a TI = 1800 ms for both readers. The optimal TI values in subjects below 50 years of age were 1600 and 1800 ms, whereas in subjects above 50 years of age, the optimal TI value was 1800 ms. The optimal TI value for the visualization of renal arteries using time-SLIP technique at 3T MRI was 1800 ms. Subjects' age affected optimal TI and this is likely due to differences in the blood velocity of the abdominal aorta.

Rhizodeposition of organic carbon by plants with contrasting traits for resource acquisition: responses to different fertility regimes

Rhizodeposition plays an important role in mediating soil nutrient availability in ecosystems. However, owing to methodological difficulties (i.e., narrow zone of soil around roots, rapid assimilation by soil microbes) fertility-induced changes in rhizodeposition remain mostly unknown. We developed a novel long-term continuous 13C labelling method to address the effects of two levels of nitrogen (N) fertilization on rhizodeposited carbon (C) by species with different nutrient acquisition strategies. Fertility-induced changes in rhizodeposition were modulated by root responses to N availability rather than by changes in soil microbial biomass. Differences among species were mostly related to plant biomass: species with higher total leaf and root biomass also had higher total rhizodeposited C, whereas species with lower root biomass had higher specific rhizodeposited C (per gram root mass). Experimental controls demonstrated that most of the biases commonly associated with this type of experiment (i.e., long-term steady-state labelling) were avoided using our methodological approach. These results suggest that the amount of rhizodeposited C from plants grown under different levels of N were driven mainly by plant biomass and root morphology rather than microbial biomass. They also underline the importance of plant characteristics (i.e., biomass allocation) as opposed to traits associated with plant resource acquisition strategies in predicting total C rhizodeposition.

Nonenhanced renal MR angiography using steady-state free precession (SSFP) and time-spatial labeling inversion pulse (Time-SLIP): repeatability and comparison of different tagging location

To prospectively determine the repeatability of noncontrast-enhanced renal arterial angiography with steady-state free precession (SSFP) and time-spatial labeling inversion pulse (Time-SLIP), and to compare the visibility of renal artery and its branches when different locations of tagging pulse were placed. Thirty-six young healthy volunteers were enrolled in this study and were twice examined by noncontrast-enhanced renal arterial angiography with SSFP and Time-SLIP in 1.5T MR scanner. Measurement error and repeatability were assessed for each of the five major parameters [vessel-to-kidney ratio (VKR), grade of renal arterial branching, grading of image quality, diameter and area of the main renal artery] using the Bland-Altman plot. Two independent observers recorded the values of the parameters; Inter- and intra-observer agreement was assessed using the intraclass correlation coefficients (ICCs). The same parameters, acquired at the tagging pulse placed just above the superior poles of both kidneys or closer to the main renal arteries, were compared using the Wilcoxon signed-rank test. Grading of arterial branching by the Time-SLIP SSFP was satisfactorily reproducible with the mean score of greater 3.83 indicating the visibility of branches within the renal parenchyma. The image quality was excellent for Segment I (the main trunk of renal artery) and good for Segment II (segmental branches pre renal parenchyma) and III (vessels within the renal parenchyma) with a satisfying repeatability between two examinations and a good inter- and intra-observer agreement. The ICCs for the inter- and intra-observer measurements of both diameter and area of the main arteries ranged from 0.781 to 0.934, indicating very good agreement. The repeatability of VKR was poor between the two examinations and at the two different tagging pulse locations. The position of tagging pulse in the origination of the main renal arteries was better than in the superior poles of kidneys as it provided a better visualization of arterial branches. Noncontrast-enhanced renal artery angiography with SSFP and Time-SLIP yields reliable and reproducible visualization of normal renal arteries. Localization of the tagging pulse closer to the main renal arteries provides better visibility of renal artery and its branches than the tag placement just above the superior poles of both kidneys.

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.

Highlighting the tricarboxylic acid cycle: Liquid and gas chromatography–mass spectrometry analyses of 13C-labeled organic acids

The tricarboxylic acid (TCA) cycle is involved in the complete oxidation of organic acids to carbon dioxide in aerobic cells. It not only uses the acetyl-CoA derived from glycolysis but also uses breakdown products of proteins, fatty acids, and nucleic acids. Therefore, the TCA cycle involves numerous carbon fluxes through central metabolism to produce reductant power and transfer the generated electrons to the aerobic electron transport system where energy is formed by oxidative phosphorylation. Although the TCA cycle plays a crucial role in aerobic organisms and tissues, the lack of direct isotopic labeling information in its intermediates (organic acids) makes the quantification of its metabolic fluxes rather approximate. This is the major technical gap that this study intended to fill. In this work, we established and validated liquid and gas chromatography–mass spectrometry methods to determine 13C labeling in organic acids involved in the TCA cycle using scheduled multiple reaction monitoring and single ion monitoring modes, respectively. Labeled samples were generated using maize embryos cultured with [13C]glucose or [13C]glutamine. Once steady-state labeling was reached, 13C-labeled organic acids were extracted and purified. When applying our mass spectrometric methods to those extracts, mass isotopomer abundances of seven major organic acids were successfully determined.

Abstract 1251: Anti-cancer mechanisms of se compounds: A stable isotope-resolved metabolomic (SIRM) approach

Abstract Recent rapid advances in analytical tools, especially NMR and mass spectrometry, allow an unprecedented range of metabolites and their transformation pathways to be determined, which transcends classical targeted metabolic studies. We have coupled these tools with stable isotopic tracers [1-3] to investigate the anticancer efficacy and mechanism(s) of different selenium forms using lung adenocarcinoma A549 cells as a model. Selenite (SeO3), methylseleninic acid (MSA) and selenomethionine (SeM) induced distinct changes in steady-state concentrations and 13C labeling patterns of various metabolites including amino & organic acids, nucleotides, antioxidants, as well as phospholipids (PL) and PL metabolites. These changes were determined using a combination of multi-nuclear 2-D NMR, GC-MS, and FT-ICR-MS techniques, which enabled perturbations in multiple metabolic pathways to be simultaneously reconstructed for relating to phenotypic changes. At low µM levels, both SeO3 and MSA inhibited growth, caused cytoskeleton disintegration, and reduced the mitochondrial redox potential in A549 cells. SeM was growth-stimulatory at ≤ 10 µM and inhibited growth but only at 40-fold higher concentration and the extent was much less than MSA or SeO3. These phenotypic differences were related to the early inhibition of glutaminolysis, Krebs cycle, and protein biosynthesis by SeO3, rapid inhibition of pyruvate carboxylation by MSA. SeM had few comparable metabolic effects. Subsequent inhibition of PL biosynthesis was also more substantial in MSA and SeO3 than in SeM-treated cells. The protein and gene expression for key enzymes in these pathways were probed to discern altered metabolic regulations. Thus, the anti-cancer action of MSA differed from that of SeO3 while SeM was not effective against lung cancer cells. The SIRM approach facilitated the resolution of the complex, form-specific anticancer mechanism of Se compounds and is generally applicable to other cancer research problems. 1. Fan, T., et al., Metabolomics-edited transcriptomics analysis of Se anticancer action in human lung cancer cells. Metabolomics Journal, 2005. 1(4): p. 325-339 2. Lane, A.N. and T.W.-M. Fan, Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY. Metabolomics, 2007. 3: p. 79-86. 3. Lane, A.N., et al., Isotopomer analysis of lipid biosynthesis by high resolution mass spectrometry and NMR. Analytica Chimica Acta, 2009. 651: p. 201-208. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2011-1251

RRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Secretory Trafficking Signal Encoded in the Carboxyl-Terminal Region of the CGβ-Subunit

Although the LHbeta- and chorionic gonadotropin-beta- (CGbeta) subunits share a high degree of sequence identity (>85%) in the first 114 amino acids, there is considerable sequence divergence at their carboxy ends. The CGbeta-subunit terminates with a unique carboxyl-terminal extension (115-145; carboxyl-terminal peptide), which contains four O-linked oligosaccharides, whereas the LHbeta-subunit bears a hydrophobic heptapeptide (115-121) at its carboxy terminus. LH is released through the regulated pathway in the pituitary, whereas CG is secreted constitutively from the placenta. We previously demonstrated in rat somatotroph-derived GH(3) cells that the LH is associated primarily with a regulated routing, and although the majority of CG was released constitutively from the cells, there was a fraction that was segregated through the regulated pathway. Moreover, we showed that the LHbeta heptapeptide is a determinant for the regulated secretion of LH. Given that the primary evolutionary change between LHbeta and CGbeta occurred at the carboxy terminus, these data suggested that the presence of the CGbeta carboxyl-terminal peptide region is responsible for the constitutive secretion of CG. A CG114 mutant (CGDeltaT) was constructed and expressed in GH(3) cells. Steady-state labeling and pulse-chase experiments demonstrated that the CGDeltaT entered the regulated pathway resulting in over 4-fold increase in the intracellular pool. The secretagogue, forskolin, stimulated CGDeltaT release over 3-fold, which was accompanied by a parallel intracellular decrease, and only marginal stimulation of CG was seen. Immunofluorescence demonstrated a unique membrane pattern of staining for CGDeltaT compared with dispersed cytoplasmic puncta for CG. Stimulation with forskolin caused a significant reduction in the relative fluorescence of CGDeltaT cells compared with a minor reduction for CG. These data show that the CGDeltaT analog resembles LH in its intracellular trafficking, further supporting the hypothesis that determinants at the carboxyl-terminal end of the CGbeta-subunit evolved from the LHbeta-subunit primarily to overcome the slow release and intracellular storage of LH resulting in rapid secretion of CG from the placenta.

Mammalian cerebral metabolism and amino acid neurotransmission during hibernation

This report demonstrates that during the torpor phase of hibernation, hamsters utilize (14)C and (13)C glucose in torpor-specific brain metabolic pathways. Microdialysis of (14)C glucose into the striatum rapidly induced a steady state labeling of extracellular fluid (ECF) lactate and labeling of tissue GABA, glutamate, glutamine, and alanine in ipsilateral and contralateral striata. The same tissue metabolites were labeled in cortex, hypothalamus, and brainstem after microdialysis of (14)C lactate into the lateral ventricle. Serine, aspartate, glycine, taurine, tyrosine, and methionine were not synthesized from glucose or lactate during torpor. ECF levels of amino and organic acids were low and unchanging during torpor and increased late during arousal to cenothermia. Labeled intracellular (14)C GABA and glutamate were not communicated to the striatal ECF or ventricular space during torpor. (13)C NMR demonstrated rapid formation of lactate and functional tricarboxylic acid cycles in GABAergic and glutamatergic neurons, and enrichment of glutamine and alanine after i.v. (13)C glucose. Large changes in tissue levels of amino acids occur prior to or during entrance into torpor but not during torpor. It is proposed that cerebral intracellular dehydration, the enlargement of ECF and the biochemistries associated with brain water homeostasis may have a role in regulating hibernation.

Reaction of fluorogenic reagents with proteins: I. Mass spectrometric characterization of the reaction with 3-(2-furoyl)quinoline-2-carboxaldehyde, Chromeo P465, and Chromeo P503

3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), Chromeo P465, and Chromeo P503 are weakly fluorescent reagents that react with primary amines to produce fluorescent products. We studied the reaction of these reagents with α-lactalbumin by mass spectrometry. The reaction generated a set of products by the addition of one or more labels to the protein. At room temperature, the reaction was an order of magnitude faster with the Chromeo reagents than with FQ; however, the steady-state labeling efficiency was a factor of two higher for FQ compared with the Chromeo reagents. The relative abundance of the products with FQ usually followed a binomial distribution, which suggests that the labeling sites were uniformly accessible to this reagent. In contrast, the distribution of reaction products with the Chromeo reagents did not follow a binomial distribution for reactions performed in the absence of sodium dodecyl sulfate (SDS); it appears that the protein labeled with the Chromeo reagents refolded into a relatively stable secondary structure that hid some reactive sites. The reaction with the Chromeo reagent did follow the binomial distribution if the protein underwent treatment with 1% SDS at 95 °C for 5 min, which apparently disrupts the protein's secondary structure and allowed uniform access to all labeling sites. Chromeo 503 labeled seven of the 13 primary amines in denatured α-lactalbumin.

Dynamics of positional enrichment: Theoretical development and application to carbon labeling in Zymomonas mobilis

Positional enrichment analysis has become an important technique for assessing detailed flux distributions and the fates of specific atoms in metabolic pathway systems. The typical approach to positional enrichment analysis is performed by supplying specifically labeled substrate to a cell system, letting the system reach steady state, and measuring where label had arrived and accumulated. The data are then evaluated mathematically with the help of a linear stoichiometric flux distribution model. While this procedure has proven to yield new and valuable insights, it does not address the transient dynamics between providing label and its ultimate steady-state distribution, which is often of great interest to the experimentalist (pulse labeling experiments). We show here that an extension of a recent mathematical method for dynamic labeling analysis is able to shed light on these transitions, thereby revealing insights not obtained with traditional positional enrichment analyses. The method traces the dynamics of one or more carbons through fully regulated metabolic pathways, which, in principle, may be arbitrarily complex. After a brief review of the earlier method and description of the theoretical extension, we illustrate the method with an analysis of the pentose phosphate pathway in Zymomonas mobilis, which has been used for traditional positional enrichment analyses in the past. We show how different labeling schemes result in distinctly different transients, which nevertheless eventually lead to a steady-state labeling profile that coincides exactly with the corresponding profile from traditional analysis. Thus, over the domain of commonality, the proposed method leads to results equivalent to those from state-of-the-art existing methods. However, these steady-state results constitute only a small portion of the insights obtainable with the proposed method. Our method can also be used as an “inverse” technique for elucidating the topology and regulation of pathway systems, if appropriate time series data are available. While such dynamic data are still rather rare, they are now being generated with increasing frequency and we believe it is desirable, and indeed necessary, to accompany this trend with an adequate, rigorous method of analysis.

A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips

In order to understand the role of sucrose synthase (SuSy) in carbon partitioning, metabolic fluxes were analyzed in maize root tips of a double mutant of SuSy genes, sh1 sus1 and the corresponding wild type, W22. [U- 14C]-glucose pulse labeling experiments permitted the quantification of unidirectional fluxes into sucrose, starch and cell wall polysaccharides. Isotopic steady-state labeling with [1- 13C]-, [2- 13C]- or [U- 13C]-glucose followed by the quantification by 1H-NMR and 13C-NMR of enrichments in carbohydrates and amino acids was also performed to determine 29 fluxes through central metabolism using computer-aided modeling. As a consequence of the suppression of SUS1 and SH1 isozymes, maize root tips diameter was significantly decreased and respiratory metabolism reduced by 30%. Our result clearly established that, in maize root tips, starch is produced from ADP-Glc synthesized in the plastid and not in the cytosol by sucrose synthase. Unexpectedly, the flux of cell wall synthesis was increased in the double mutant. This observation indicates that, in maize root tips, SH1 and SUS1 are not specific providers for cellulose biosynthesis.

Selective retention and degradation of molecules with a single mutant α1(I) chain in the Brtl IV mouse model of OI

We investigated the secretion, matrix incorporation and interactions of molecules with one and two mutant α1(I) collagen chains in the Brtl IV murine model for Osteogenesis Imperfecta, carrying a Gly-349 to Cys substitution in one col1a1 allele. We detected a significant deviation from the expected 25 and 50% content of the molecules with no (37–46%) and one (26–40%) mutant chains in skin and bone as well as in fibroblast and osteoblast cell culture media. Steady-state labeling with 35S-Cys demonstrated incomplete secretion of the mutant collagen in cell culture, particularly molecules containing one mutant chain. Pulse and pulse-chase experiments revealed slower secretion of the latter. An enlargement of endoplasmic reticulum in skin fibroblasts from Brtl IV mice, clearly visible by electron microscopy, supported the abnormal secretion identified by biochemical studies. We observed increased susceptibility of molecules with one mutant chain to proteolytic degradation in vitro, but we did not detect significant selective degradation in cell culture media. Mutant collagen molecules incorporated from the media into newly deposited fibers and into fully crosslinked and mature matrix in the same ratio as they were secreted. Specific labeling of reactive –SH demonstrated that about half of the Cys349–SH groups in the mutant molecules were exposed and potentially available for aberrant interactions with other molecules inside or outside the cells. Based on these and our previous findings, we argue that the outcome in Brtl IV may be significantly affected by cellular stress and malfunction caused by the retention and degradation of newly synthesized mutant collagen.

Substrate cycles in the central metabolism of maize root tips under hypoxia

Substrate cycles, also called “futile” cycles, are ubiquitous and lead to a net consumption of ATP which, in the normoxic maize root, have been estimated at about 50% of the total ATP produced [Alonso, A.P., Vigeolas, H., Raymond, P., Rolin, D., Dieuaide-Noubhani, M., 2005. A new substrate cycle in plants. Evidence for a high glucose-phosphate-to-glucose turnover from in vivo steady-state and pulse-labeling experiments with [ 13C] glucose and [ 14C] glucose. Plant Physiol. 138, 2220–2232]. To evaluate their role we studied the substrate cycles of maize root tips under an oxygen limitation of respiration (3% O 2). Short-time labeling experiments with [U- 14C]-Glc were performed to quantify the fluxes through sucrose and starch cycles of synthesis and degradation. Steady-state labeling with [1- 13C]-Glc followed by 1H NMR and 13C NMR analysis of sugars and free alanine was used to quantify fluxes in the central metabolic pathways, including the Glc-P/Glc cycle and the fructose-P/triose-P cycle of glycolysis. Comparison with results previously obtained in normoxia [Alonso et al., as mentioned above] showed that 3% O 2 induced fermentation and reduced respiration, which led to a lesser amount of ATP produced. The rates of Glc consumption, glycolytic flux and all substrate cycles were lower, but the proportion of ATP consumed in the substrate cycles remained unchanged. These findings suggest that substrate cycles are not a luxury but an integral part of the organization of the plant central metabolism.