Citrate Synthesis Research Articles

Metabolic profiling of retrograde pathway transcription factors rtg1 and rtg3 knockout yeast.

Rtg1 and Rtg3 are two basic helix-loop-helix (bHLH) transcription factors found in yeast Saccharomyces cerevisiae that are involved in the regulation of the mitochondrial retrograde (RTG) pathway. Under RTG response, anaplerotic synthesis of citrate is activated, consequently maintaining the supply of important precursors necessary for amino acid and nucleotide synthesis. Although the roles of Rtg1 and Rtg3 in TCA and glyoxylate cycles have been extensively reported, the investigation of other metabolic pathways has been lacking. Characteristic dimer formation in bHLH proteins, which allows for combinatorial gene expression, and the link between RTG and other regulatory pathways suggest more complex metabolic signaling involved in Rtg1/Rtg3 regulation. In this study, using a metabolomics approach, we examined metabolic alteration following RTG1 and RTG3 deletion. We found that apart from TCA and glyoxylate cycles, which have been previously reported, polyamine biosynthesis and other amino acid metabolism were significantly altered in RTG-deficient strains. We revealed that metabolic alterations occurred at various metabolic sites and that these changes relate to different growth phases, but the difference can be detected even at the mid-exponential phase, when mitochondrial function is repressed. Moreover, the effect of metabolic rearrangements can be seen through the chronological lifespan (CLS) measurement, where we confirmed the role of the RTG pathway in extending the yeast lifespan. Through a comprehensive metabolic profiling, we were able to explore metabolic phenotypes previously unidentified by other means and illustrate the possible correlations of Rtg1 and Rtg3 in different pathways.

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet – A pilot study

Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake.

A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves.

Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis.

Effect of gold nanoparticles on the photocatalytic efficiency of ZnO films

This study presents a simple and inexpensive method for preparation of ZnO/Au thin film photocatalysts. Nanostructured ZnO and modified with gold nanoparticles zinc oxide films are obtained on glass substrate by dip-coating technique. The precursor suspension is prepared using commercial ZnO powder, ethanol and polyethyleneglycol, while the gold nanoparticles are obtained by classical citrate synthesis. Thin ZnO and ZnO/Au films are characterized by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), X-ray diffraction and UV-Vis spectroscopy. The photocatalytic action of the samples is studied in respect to Malachite Green photodegradation under UV and visible light irradiation. The modified ZnO/Au films manifest higher photocatalytic efficiency under UV and visible light illumination in comparison to ZnO ones. A possible mechanism for explanation of the observed effect is proposed.

Characterization of Nickel Ions in Nickel‐Doped Yttria‐Stabilized Zirconia

The distribution of Ni2+ ions in NiO‐doped 10YSZ powder is examined with Superconducting Quantum Interference Device magnetometry, a technique that is able to distinguish between randomly distributed Ni2+ ions in solid solution and ordered Ni2+ ions within NiO with high precision. Very high purity powders containing 0.01, 0.1, 0.5, and 1.0 mol% NiO in 10YSZ (all levels below the solid solubility limit of NiO in 10YSZ) were made from acetate precursors and a modified EDTA (ethylenediaminetetraacetic acid)‐citrate synthesis method. The powders were calcined in air at either 873 or 1273 K. The 873 K calcination leads to single phase YSZ particles about 10 nm in diameter, and almost all of the NiO dopant exists in complete solid solution. The 1273 K calcination leads to a larger YSZ particle size (55–95 nm), and also to the formation and/or growth of NiO particles, the amount of which depends on the length of time of calcination. Upon sintering the powders in air (1773 K, 1 h), the NiO dissolves back into 10YSZ. The results demonstrate that particle growth during calcination leads to the exsolution of Ni2+ ions to form NiO. This has important implications for the synthesis of NiO‐doped 10YSZ from chemical precursors.

Metabolic alterations due to IDH1 mutation in glioma: opening for therapeutic opportunities?

Recently heterozygous mutations in the active site of the enzyme isocitrate dehydrogenase 1 (IDH1) were discovered in glioblastomas [1]. In cohorts of glioma patients the IDH1 mutation appeared to be a strong predictor of clinical outcome, overruling histological malignancy grade [2]. IDH1 is an enzyme of the tricarboxylic acid (TCA) cycle and is located in the cytosol, where it produces NADPH by transforming isocitrate into α-ketoglutarate. Because the mutant enzyme displays neomorphic activity through NADPH-dependent transformation of α-ketoglutarate into 2-hydroxyglutarate (2HG), the tumorigenic role of the increased levels of 2HG has become a target of speculation [3]. IDH1 mutation alters the cellular metabolism and epigenetic phenotype influencing cellular proliferation. IDH1 mutation infers increased levels of D2HGDH leading to the inhibition of DNA and histone demethylating enzymes, resulting in the glioma-CpG island phenotype [4]. Altered concentrations of pyruvate kinase M2 play also a role in histone modifications which are associated with the transcription of the proliferation-related cyclin D1 and c-MYC [5]. In addition, IDH1 mutant cells show alterations in glutamine, fatty acid and citrate synthesis pathways, which all may have their influence on cellular proliferation [5]. The changes in IDH1 function affect the glucose metabolism, which may explain the different biological behaviour of tumors with and without the IDH1 mutation [6]. In order to detect these changes, we compared the expression levels of the genes participating in the TCA cycle and in the anaerobic glycolysis in 33 IDH1 mutated samples (3 astrocytomas WHO grade II; 6 astrocytomas WHO grade III; 4 glioblastomas; 9 oligodendrogliomas WHO grade II; 11 oligodendrogliomas WHO grade III) and in 39 IDH1 wild-type glioma samples (10 astrocytomas WHO grade III; 26 glioblastomas; 1 oligodendroglioma WHO grade II; 2 oligodendroglioma WHO grade III) and in four samples of normal brain (Table 1). We found expressional differences of 16/24 genes (Figure 1). The IDH1 mutated cells seem to compensate for the low production of α-ketoglutarate by overexpressing D2HGDH and L2HGDH in the cytoplasm. They also overexpress GLUD1, which converts glutamate to α-ketoglutarate inside the mitochondria. In addition, we found that IDH1 mutated cells overexpress HIF1AN. The HIF1AN gene inhibits HIF1α. Since HIF1α acts as an oxygen sensor that promotes angiogenesis, the formation of dysfunctional tumor vasculature is counteracted in IDH1 mutated cells. Furthermore, the IDH1 mutated cells overexpress the LDHB gene while cells without IDH1 mutation overexpress the LDHA gene. The present results illustrate that tumor cells without IDH1 mutation switch their energy production from a low rate of glycolysis followed by the TCA cycle to a high rate of glycolysis followed by aerobic glycolysis (LDHA up-regulation; Figure 1). The resulting lactate acid production causes tissue acidosis known as the Warburg effect. In invasive cancers, the pH of the extracellular space increases the infiltrative potential of the tumor cells [7]. In addition, normalization of the extracellular pH by alterations of the enzymatic actions of LDHA and LDHB influences the progression of cancer cells [8,9]. It may well be that glial tumor cells with IDH1 mutation tend to correct their energy production through the TCA cycle by overexpressing LDHB (Figure 1). By doing so, they normalize the tissue pH which offers yet another explanation for the less aggressive biological behavior of the IDH1 mutated gliomas. Table 1 Percentages of glioma types and grades Figure 1 Overview of alterations in glucose metabolism in the IDH1 mutated and IDH1 wild-type gliomas relative to glucose metabolism in normal brain. The enzymes included in the analysis are shown in black. The bar diagrams show the averaged expressional levels ... We conclude that gliomas with IDH1 mutation normalize their glucose metabolism, which appears to result in a slower tumor progression. Depending on the IDH1 status of the tumor, specific interference with the glucose metabolism and aerobic glycolysis should therefore be considered for future therapeutic strategies.

Raman probing trace melamine in milk by a functionalized test paper

The rapid sensing for melamine in milk products has been developed with a portable Raman system based on the facile synthesis of tri-sodium citrate and dodecasodium of phytic acid (IP6) dual-functionalized gold nanoparticles modified filter paper, shortly named IP6-TC@Au NPs test paper. The tri-sodium citrate improved the sensitivity of Raman probe and the later kept the shelf stability. The limit of detection for melamine in water is 5μM (0.63ppm) and it is 10μM (1.26ppm) for testing real milk sample, which is well below the FDA's tolerance level of 2.5ppm in milk. The proposed easy assay of IP6-TC@Au NPs test paper combining with portable Raman system could be applied for on-site monitoring melamine in milk products.

The alternative respiratory pathway mediates carboxylate synthesis in white lupin cluster roots under phosphorus deprivation

Plant adaptations associated with a high efficiency of phosphorus (P) acquisition can be used to increase productivity and sustainability in a world with a growing population and decreasing rock phosphate reserves. White lupin (Lupinus albus) produces cluster roots that release carboxylates to efficiently mobilize P from P-sorbing soils. It has been hypothesized that an increase in the activity of the alternative oxidase (AOX) would allow for the mitochondrial oxidation of NAD(P)H produced during citrate synthesis in cluster roots at a developmental stage when there is a low demand for ATP. We used the oxygen-isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in different root sections of white lupins grown hydroponically with and without P. In parallel, AOX protein levels and internal carboxylate concentrations were determined in cluster and non-cluster roots. Higher in vivo AOP activity was measured in cluster roots when malate and citrate concentrations were also high, thus confirming our hypothesis. AOX protein levels were not always correlated with in vivo AOP activity, suggesting post-translational regulation of AOX.

Fast and simple one-step preparation of 68Ga citrate for routine clinical PET

The imaging of infectious and inflammatory diseases using gallium-67 (⁶⁷Ga) citrate scintigraphy has been a well-established diagnostic tool for decades. In recent times, interest has focused on PET using the short-lived positron emitting radioisotope ⁶⁸Ga. ⁶⁸Ga is not only more readily available, it also provides better quality images whose high resolution permits quantitative analyses, thus improving the management of patients suffering from infections or inflammation. The purpose of our study was to develop a fast and reliable synthesis protocol for the preparation of ⁶⁸Ga citrate under good manufacturing practice aspects without the use of organic solvents. A commercially available synthesis module was used to perform 10 syntheses with an average yield of 768 ± 31 MBq (mean ± SD) within 10 min; 92.04 ± 1.23% of the radioactivity was located in the product vial, and the rest on the cation exchange cartridge (7.48 ± 1.23%) and in the waste vial (0.47 ± 0.28%). The radiochemical purity of the product determined by instant thin-layer chromatography was greater than 99%. The products have been proven to be sterile and pyrogen-free. Variations were made in several critical synthesis parameters, and the results are presented herein. By eliminating the use of organic solvents, the previously required quality control testing of the final product by gas chromatography can be abandoned. This novel, high-yielding method allows for a more efficient synthesis of ⁶⁸Ga citrate with both shorter production time and high radiochemical purity.

A combination of five mechanisms confers a high tolerance for aluminum to a wild species of Poaceae, Andropogon virginicus L.

How can high tolerance against aluminum (Al) toxicity be obtained in plants? To address this question, tolerant mechanisms were characterized in a highly Al tolerant wild species of Poaceae, Andropogon virginicus L. A. virginicus showed an Al-stress-induced synthesis and secretion of citrate and malate in roots. This mechanism may help to suppress an increase of toxic Al ions in the root region. Microscopic observation of the morin-stained leaves indicated that the Al transferred to shoots was specifically accumulated in the trichomes and spikes of the leaves and that some portion of the accumulated Al was furthermore secreted as sap from the tips of trichomes. Al-induced synthesis of poly-phenolic compounds including anthocyanin also occurred in roots as a long term response to Al toxicity and anthocyanin production did not co-localize with either Al accumulation, nitric oxide (NO) production or lipid peroxides production in the roots. It was suggested that oxidative damage caused by Al stress was suppressed in these areas where anthocyanin was localized. Moreover, induction of NO production occurred in roots within 24h of Al treatment. Our results suggested that NO could not efficiently ameliorate the Al-dependent nuclei deformation and DNA fragmentation, but could function as a trigger to stimulate anti-peroxidation enzymes under Al stress. Collectively the results suggested that A. virginicus manifests its high Al tolerance by a unique combination of effective mechanisms.

Implementing atomic force microscopy (AFM) for studying kinetics of gold nanoparticle's growth

In a novel experimental approach Atomic Force Microscopy (AFM) was applied as a tool for studying the kinetics of gold nanoparticle growth. The gold nanoparticles were obtained by classical Turkevich citrate synthesis at two different temperatures. From the analysis of AFM images during the synthesis process the nanoparticle s’ sizes were obtained. To demonstrate the applicability and the reliability of the proposed experimental approach we studied the nanoparticles growth at two different temperatures by spectrophotometric measurements and compared them with the results from AFM experimental approach. We also compared AFM experimental data with Dynamic Light Scattering (DLS) and with Transmission Electron Microscopy (TEM) data. The experimental data from all the applied methods were fitted with two step Finke–Watzky kinetics model and the corresponding kinetics constants were obtained and compared.

Cofactor Balance by Nicotinamide Nucleotide Transhydrogenase (NNT) Coordinates Reductive Carboxylation and Glucose Catabolism in the Tricarboxylic Acid (TCA) Cycle

Cancer and proliferating cells exhibit an increased demand for glutamine-derived carbons to support anabolic processes. In addition, reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was recently shown to be a major source of citrate synthesis from glutamine. The role of NAD(P)H/NAD(P)(+) cofactors in coordinating glucose and glutamine utilization in the tricarboxylic acid (TCA) cycle is not well understood, with the source(s) of NADPH for the reductive carboxylation reaction remaining unexplored. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial enzyme that transfers reducing equivalents from NADH to NADPH. Here, we show that knockdown of NNT inhibits the contribution of glutamine to the TCA cycle and activates glucose catabolism in SkMel5 melanoma cells. The increase in glucose oxidation partially occurred through pyruvate carboxylase and rendered NNT knockdown cells more sensitive to glucose deprivation. Importantly, knocking down NNT inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma cells. Overexpression of NNT is sufficient to stimulate glutamine oxidation and reductive carboxylation, whereas it inhibits glucose catabolism in the TCA cycle. These observations are supported by an impairment of the NAD(P)H/NAD(P)(+) ratios. Our findings underscore the role of NNT in regulating central carbon metabolism via redox balance, calling for other mechanisms that coordinate substrate preference to maintain a functional TCA cycle.

Synthesis and thermal stability of Re2Zr2O7, (Re=La, Gd) and La2(Zr1−xCex)2O7−δ compounds under reducing and oxidant atmospheres for thermal barrier coatings

Rare-earth zirconates and cerates have attracted particular interest for thermal barrier coating (TBC) applications due to their advantageous thermal properties, such as a low conductivity and efficient phase stability at elevated temperatures. This study focuses on synthesising La 2 Zr 2 O 7 , Gd 2 Zr 2 O 7 , La 2 Ce 2 O 7− γ and La 2 (Zr 0.7 Ce 0.3 )O 7− γ compounds via two soft chemistry processes, alkoxide and citrate synthesis. Thermal analysis, X-ray diffraction (XRD) and scanning electron microscope observations were used to analyse the powder after calcinations under air. Chemical reactivity tests under a reducing atmosphere were performed at 1400 °C and investigated by XRD analysis. It was found that the lanthanum and gadolinium zirconates are the most stable and interesting materials under an Ar (g) /3%H 2(g) atmosphere.

Enhanced photocatalytic hydrogen generation from barium tantalate composites

(111)-layered Ba5Ta4O15 photocatalysts were synthesised by a solid state reaction route and a citrate synthesis route, and their structural and electronic properties were investigated. After citrate route preparation, the presence of a second phase, namely Ba3Ta5O15, was determined by X-ray powder diffraction and absorption spectroscopy. The existence of this phase had a profound effect on the photocatalytic activity of this Ba5Ta4O15/Ba3Ta5O15 composite in comparison to the pure Ba5Ta4O15 materials. The photocatalytic performance of the barium tantalates was evaluated by investigating the capability in ˙OH radical formation and hydrogen generation. Strongly increased hydrogen evolution rates for the Ba5Ta4O15/Ba3Ta5O15 composite, up to 160% higher than for the pure Ba5Ta4O15, were determined, and only very small amounts of Rh co-catalyst, deposited on the photocatalysts by stepwise reductive photo-deposition, were needed to achieve these results.

Ambient temperature synthesis of citrate stabilized and biofunctionalized, fluorescent calcium fluoride nanocrystals for targeted labeling of cancer cells.

Targeted biological contrast agents are emerging as promising candidates in the field of cancer theragnostics. Herein, we report an ambient temperature synthesis of a nanosized, antibody functionalized lanthanide doped CaF2 biolabel and demonstrate in vitro its potential for cancer cell targeting efficacy and specificity. Monodispersed citrate stabilized lanthanide (Eu3+) doped CaF2 nanoparticles with size ∼25 nm, exhibiting strong fluorescent emission at 612 nm, were prepared using an aqueous wet chemical route at room temperature. Biofunctionalization of the fluorescent nanoparticles using an anti-EGFR antibody through EDC-NHS coupling chemistry enabled targeting of EGFR over-expressing cells. The nanobioconjugates showed preferential binding to EGFR+ve oral epithelial carcinoma cells (KB) and human epidermoid carcinoma cells (A431) with no accumulation onto EGFR-ve non-cancerous NIH 3T3 cells. The fluorescence was maintained after the bioconjugation as well as after attachment to the cancer cells, demonstrating their potential as targeted biolabels. Cytotoxicity evaluation with several cancerous (A431, KB) and non-cancerous (NIH 3T3, L929) cell lines revealed no toxicity at concentrations up to 1 mM. Thus, the fluorescence characteristics and biocompatibility, coupled with the molecular receptor targeting capability, suggest the potential use of CaF2 in the field of bioimaging.

The significant effect of the phase composition on the oxygen reduction reaction activity of a layered oxide cathode

Layered oxides of Sr4Fe4Co2O13 (SFC2) which contains alternating perovskite oxide octahedral and polyhedral oxide double layers are attractive for their mixed ionic and electronic conducting and oxygen reduction reaction properties. In this work, we used the EDTA–citrate synthesis technique to prepare SFC2 and vary the calcination temperature between 900 and 1100 °C to obtain SFC2, containing different phase content of perovskite (denoted as SFC-P) and (Fe,Co) layered oxide phases (SFC-L). Rietveld refinements show that the SFC-P phase content increased from ∼39 wt% to ∼50 wt% and ∼61 wt% as the calcination temperature increased from 900 °C (SFC2-900) to 1000 °C (SFC2-1000) and 1050 °C (SFC2-1050). At 1100 °C (SFC2-1100), SFC-P became the dominant phase. The oxygen transport properties (e.g. oxygen chemical diffusion coefficient and oxygen permeability), electrical conductivity and oxygen reduction reaction activity is enhanced in the order of SFC2-1000, SFC2-1100 and SFC2-1050. The trend established here therefore negates the hypothesis that the perovskite phase content correlates with the oxygen transport property enhancement. The results suggest instead that there is an optimum composition value (e.g. 61 wt% of SFC-L for SFC2-1050 in this work) on which synergistic effects take place between the SFC-P and SFC-L phase.

Synthesis and Characterization of Citrate Coated Magnetite Nanoparticles

Citrate coated magnetite nanoparticles (NPs) were prepared by a modified coprecipitation technique in the presence of sodium citrate and sodium acetate. The citrate coated NPs showed a close particle size distribution, superparamagnetic behavior, and single crystalline nature. Water dispersions of magnetite NPs were analyzed by laser diffraction; this technique revealed that the NPs form micrometer sized aggregates. It was found that the size of the aggregates depends strongly on the time of ultrasonic agitation and the citrate shell.

Dielectric properties of hexagonal perovskite ceramics prepared by different routes

In this work, four different methods, i.e. solid state reaction, citrate sol–gel process, Pechini and microwave are employed to synthesize Ba4YMn3O11.5±δ ceramics. The phase structure of the powders can be well indexed as a 12R hexagonal perovskite crystallizing in space group R3¯m. The density and morphology (average grain size) of sintered samples vary with the synthesis processes. The dielectric permittivity and loss tangent of the samples have been measured in the frequency range 1kHz–1MHz. The results show that they are very sensitive to the synthesis process. The best properties are obtained for the sample synthetized by citrate process. This compound has a high dielectric permittivity (ε′r=104), which is almost frequency independent over the 100Hz–100kHz range from room temperature to 150°C. This has been attributed to the IBLC mechanism. By impedance spectroscopy analysis, all the compounds were found to be electrically heterogeneous, exhibiting semiconducting grains and insulating grain boundaries. Finally, we show that the IBLC model is well adapted to the materials obtained by the modified citrate synthesis route, but not to the ones from the other routes.

Dual effects of transgenic Brassica napus overexpressing CS gene on tolerances to aluminum toxicity and phosphorus deficiency

Low phosphorus (P) bioavailability and aluminum (Al) toxicity are two major constraints to plant growth in acid soil. To improve the tolerance of Brassica napus to Al toxicity and P deficiency, we generated transgenic canola (Brassica napus cv Westar) lines overexpressing a Pseudomonas aeruginosa citrate synthase (CS) gene and then investigated the effects of CS gene overexpressing in canola on enhancing tolerance to the two constraints. The vector construction and plant transformation, molecular identification, estimation of extracellular and cellular citrate and malate concentrations, enzyme activity and gene expression analyse and Al tolerance and P acquisition assays were conducted using both hydroponics and soil culturing in the study. Both the root citrate and malate concentrations and their exudations in the two transgenic lines significantly increased compared with wild type (WT) following exposure to Al. These increases may be attributed to higher activities of the CS, malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes in the TCA cycle and the expression of BnALMT and BnMATE in the transgenic plants following Al exposure. The primary root elongation and prolonged Al treatment (10 days) experiments revealed that the transgenic lines displayed enhanced levels of Al tolerance. In addition, they showed enhanced citrate and malate exudation when grown in P-deficient conditions. Moreover, the enzyme activities of the transgenic lines were significantly higher compared with WT in response to P-deficient stress. The soil culture experiment showed that the transgenic lines possessed improved P uptake from the soil and accumulated more P in their shoots and seeds when FePO4 was used as the sole P source. These results indicate that the overexpression of the CS gene in B. napus not only leads to increased citrate synthesis and exudation but also changes malate metabolism, which confers improved tolerances to Al toxicity and P deficiency in the transgenic plants. These findings provide further insight into the dual effects of CS gene overexpression on Al toxicity and P deficiency in plants.

Measurement of Acetyl‐CoA turnover ( ≈citric acid cycle flux) in perfused rat hearts by isotope dilution

We developed an isotope dilution technique to assess acetyl‐CoA turnover (≈ citric acid flux) in perfused rat hearts. In hearts, almost all acetate is used in citrate synthesis. Heart is perfused with buffer containing a low concentration of [2H3‐13C2] acetate (0.03 mM) which forms M5 and M4 acetyl‐CoA. The M4 acetyl‐CoA (greater than the small M4 component of M5 acetate) results probably from reversible enolization of acetyl‐CoA with partial 2H loss. The buffer may also contain one labeled substrate which generates M2 acetyl‐CoA ([13C6] glucose or [1,2‐13C2] palmitate). Total acetyl‐CoA turnover is calculated from (i) the M5 acetate uptake, and (ii) the M5 acetyl‐CoA enrichment + the increase in M4 enrichment of acetyl‐CoA compared with the small M4 enrichment of [2H3‐13C2] acetate. The contribution of glucose or palmitate to acetyl‐CoA is calculated from the M2 enrichment of acetyl‐CoA. Loss of label between M5 acetate and citrate synthesis (acetylcarnitine, ketone bodies) is negligible. Rates of acetyl‐CoA turnover calculated by our method are similar to those calculated from O2 uptake. The method was applied to measurements of acetyl‐CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). Supported by NIDDK RoadMap and the Case Mouse Metabolic Phenotyping Center.