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In human cells, cytosolic citrate is a chief precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. Cytosolic citrate further regulates the energy balance of the cell [...]

Nanocrystals of apatitic calcium phosphate impart the organic-inorganic nanocomposite in bone with favorable mechanical properties. So far, the factors preventing crystal growth beyond the favorable thickness of ca. 3 nm have not been identified. Here we show that the apatite surfaces are studded with strongly bound citrate molecules, whose signals have been identified unambiguously by multinuclear magnetic resonance (NMR) analysis. NMR reveals that bound citrate accounts for 5.5 wt% of the organic matter in bone and covers apatite at a density of about I molecule per [(2 nm).sup.2], with its three carboxylate groups at distances of 0.3 to 0.45 nm from the apatite surface. Bound citrate is highly conserved, being found in fish, avian, and mammalian bone, which indicates its critical role in interfering with crystal thickening and stabilizing the apatite nanocrystals in bone. nanocrystal stabilization | nanocomposite | organic-inorganic interface | bone composition | solid-state NMR doi/ 10.1073/pnas.1009219107

The nature of the chemical interactions between organic species and mineral surfaces and interlayers has significant implications for many geochemical processes and for medical and materials applications, but the molecular-scale characteristics of such interactions are poorly understood. Here we describe an experimental investigation of citrate-hydrotalcite (HT) that is designed to investigate the structural environments and dynamical behavior of carboxylic species interacting with the protonated surfaces of positively charged materials such as layered double hydroxides (LDHs). X-ray diffraction, compositional analysis, and thermal analysis confirm successful intercalation of trivalent citrate anions into the interlayers of the Mg/Al hydrotalcite using an ion-exchange method. The basal spacing changes from 8.9 [Angstrom] at relative humidity (R.H.) = 0% to 11.3 [Angstrom] at R.H. = 75%, to 18.9 [Angstrom] at 100% R.H., consistent with reorientation of the citrate from parallel to the hydroxide layers at low water contents to perpendicular to them at higher water contents. At the largest water contents, the citrate may lose contact with one of the hydroxide layers, in contrast to the behavior of LDHs containing small, inorganic species. The similarity of the [sup.13]C nuclear magnetic resonance (NMR) chemical shifts for citrate-HT and sodium citrate solution demonstrates the absence of grafting (covalent bond formation) between the citrate molecules and the metal hydroxide layers, and the dominance of Coulombic and H-bond interactions in stabilizing the structure. Decreasing [sup.13]C NMR peak widths, decreasing signal/noise ratios in the [sup.1]H-[sup.13]C CPMAS NMR spectra, and increasing basal spacing with increasing R.H. are consistent with increasing dynamical disorder of the interlayer citrate sites in a progressively more water-rich interlayer. Keywords: Citrate- HT, ion-exchange, XRD, thermal analysis, [sup.1]H-[sup.13]C CPMAS NMR

Excretion of organic acids, e.g. citrate, by anamorphic fungi is a frequent phenomenon in natural habitats and in laboratory cultures. In biotechnological processes for citrate production with Aspergillus niger extracellular citrate concentrations up to 1 mol [l.sup.-1] are achieved. Intracellular citrate concentrations are in the millimolar range. Therefore the question arises whether citrate excretion depends on active transport. In this article thermodynamic calculations are presented for citrate excretion by A. niger at an extracellular pH of 3 and by Penicillium simplicissimum at an extracellular pH of 7. From the results of these calculations it is concluded that in both cases a passive transport step suffices for citrate excretion.

Adsorption of zinc to two soils was quantified in the presence of citrate ranging in concentration from 0 to 10 mM/L. For both soils, Zn adsorption increased with increasing citrate up to a concentration of 2mM/L. With increasing citrate concentrations between 2 and 10 mM/L, Zn adsorption increased in one soil (pH 4.8) but decreased in the second (pH 5.8). Adsorption of a Zn-citrate complex is key to the Zn adsorption in both soils. The soil with the higher pH appears to have a significantly lower adsorption capacity for the complex than the soil with pH 4.8. Activity-based adsorption modeling supports these experimental observations. CE Database subject headings: Contamination; Heavy metals; Adsorption; Soil pollution; Zinc.

Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.

Byline: L. F. Naumenko (1), N. M. Buneeva (2), R. N. Korneeva (2), V. F. Selemenev (2), D. V. Nemtsev (3) Abstract: A simple and rapid procedure was developed for the potentiometric titration of citrate ions in fermented solutions from citric acid production with the use of a copper-selective electrode. Author Affiliation: (1) Department of Chemistry, Voronezh State University, Universitetskaya pl. 1, Voronezh, 394006, Russia (2) Voronezh State Agrarian University, ul. Michurina 1, Voronezh, 394087, Russia (3) Belgorod Citric Acid Production Plant, ul. Chicherina 8, Belgorod, 308015, Russia Article History: Registration Date: 06/10/2004

Research has been conducted on the Leuconostoc paramesenteroides citMCDEFGRP operon. The expression pattern of this operon in response to the citrate addition to the growth medium has been investigated.

Research has been conducted on the ferric citrate transport gene transcription in Escherichia coli. The control of this transcription by the signal transduction mechanism is described.

Research has been conducted on the citrate and molybdenum(VI) aqueous coordination chemistry. Results indicate the isolation of molybdenum(VI) citrato monomeric raceme and dimer.

A green fluorescent protein-expressing Escherichia coli O157:H7 strain has been used to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10 degrees Celsius. Effects of sodium salts of lactate, diacetate and buffered citrate were and combinations were studied. The pathogen was resistant to salts tested. It was confirmed that refrigerated meat contaminated with the pathogen does not stop being hazardous.

Isolation of the gltA gene that encodes Sinorhizobium meliloti has been carried out. The results of the liquid chromatography analysis of the succinylated succinoglycan are discussed.

Information about the capping action of citrate in relation to colloidal silver sols has been obtained by investigating the size and size distribution of the silver particles created at various citrate concentrations. Their absorption spectra was also assessed.

The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively. The binding and translocation properties of CitP and MleP for a broad array of substrates and substrate analogues were examined. Results were examined in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.

Research was conducted to examine the mechanism of citrate metabolism in Lactococcus lactis that provides resistance against lactate toxicity at low pH. The nucleotide sequences of the genes coding for the CitPs of L. mesenteroides and L. lactis were almost identical. Results indicate that citrate metabolism in L. lactis may produce metabolic energy comparable to that of L. mesenteroides. Findings also suggest that induced citrate metabolic pathway makes cells more resistant to the inhibitory effects of the fermentation product that accumulates under these conditions.

A ion adsoption model has been developed to describe the competitive interaction between phosphate and citrate on goethite. The presence of phosphate greatly reduced citrate adsorption over a wide pH range. Citrate affected phosphate adsorption at pH values below 7, with the largest interaction around pH 5. Competition for binding sites was the main reason for the competitive adsorption of phosphate and citrate.

The effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction are presented. Data indicate that the residues are involved in the stabilization of one or more of the transition states in the multistep citrate synthase reaction. However, none of these mutants can activate the carbonyl of oxaloacetate by polarization. All changes have effects on more than one step in the catalytic cycle. Surprisingly, the energetics of citrate synthase are tightly coupled.

Nanocrystalline [Gamma][Prime]-[Fe.sub.4]n and [Epsilon]-[Fe.sub.3]N nitrides are synthesized by using a citrate precursor route. The nitridation of [Alpha]-[Fe.sub.2][O.sub.3] results in a nitrogen deficient perovskite [Gamma][Prime]-[Fe.sub.4]N phase at 773 K and hexagonal [Epsilon]-[Fe.sub.3]N phase at 823 K. The nitride particle is a magnetic cluster consisting of an assembly of crystallites. [Gamma][Prime]-[Fe.sub.4]N is a weak itinerant ferromagnet and the particles exhibit acicular platelet-like morphology. The magnetic moments in ultrafine [Gamma][Prime]-[Fe.sub.4]N and [Epsilon]-[Fe.sub.3]N nitride particles are due to spin pairing effects, lattice expansion due to interstitial nitrogens. superparamagnetic behavior, and randomly canted spin structures at the surface. The reduction of magnetization and Curie temperature ([T.sub.c]) is attributed to fine particle size effects. In [Gamma]-[Fe.sub.4]N and [Epsilon]-[Fe.sub.3]N, the intermixing of N-2p states with Fe-3d states occurs because of similarities in their density of states, and thus the reduction of unpaired d-electrons results in the lowering of magnetic moments compared to [Alpha]-Fe. The Mossbauer study of [Gamma][Prime][Fe.sub.4]N and [Epsilon]-[Fe.sub.3]N nitrides indicate randomly canted spin structures at the surface and corroborates the observed magnetic properties. Index Terms - Ferromagnetism, iron nitride, nanocrystalline, precursor, superparamagnetism.

A radioactive labeling nuclear magnetic resonance technique has been developed to estimate relative cardiac anaplerosis using [13C] glutamate. The technique is not impaired by isotopic biochemical precursor pyruvic acid sources. Mathematical analysis and physiological pathways involving pyruvate and the citric acid cycle are illustrated. The effects of biochemicals malate-aspartate shuttle are assessed.

Research was conducted to examine a citB null mutant of Bacillus subtilis. Plasmid DNA was determined from Escherichia coli while heat resistance was measured after the end of the exponential phase for all cultures. Results indicated that the null mutant was characterized by a severe early defect in sporulation when prepared in nutrient broth sporulation medium. They also suggested that the relationship between citrate accumulation and the early block in sporulation may be related to the capability of citrate to chelate divalent cations.

The crystal structure of the closed form of citrate synthase from the hyperthermophilic archaeon Pyrococcus furiosus at 1.9 A resolution was examined. The recombinant P. furiosus was purified and crystallized. Data were later on collected on a MarResearch image plate. Results of the research showed that the increased number of intersubunit ion pairs is accompanied by an increase in number involved in networks. Furthermore, there is an increased compactness of the protein with an increase in operating temperature.

Research was conducted to show the isolation and molecular characterization of the cDNA encoding a Xenopus intestine Na+-dicarboxylate cotransporter, NaDC-2, by functional expression cloning in Xenopus oocytes. NaDC-2 encodes a 622-residue polypeptide with a predicted mass of 68.6 kDa. The transport of succinate by NaDC-2 is insensitive to pH, whereas the transport of citrate is inhibited at high pH. The differences in functional properties between NaDC-2 and the structurally related Na+-dicarboxylate cotransporters NaDC-1 and hNaDC-1 will form the basis of detailed studies.

The citrate metabolic pathway of Leuconostoc mesenteroides up to pyruvate was investigated. Experiments on the control of expression of the citrate transporter, citrate lyase and oxaloacetate decarboxylase by citrate in the growth medium indicated a branching of the pathway at the level of oxaloacetate towards the production of aspartate via transamination. It was also observed that amino acid synthesis by transamination is a more general phenomenon in Leuconostoc species and that the alpha-ketocarboxylate precursors are readily taken up from the medium by a proton motive force-driven transporter(s) in the cytoplasmic membrane.

The gene coding for the secondary transporter called CitM which mediates citrate uptake in Bacillus subtilis was cloned, sequenced and functionally expressed in Escherichia coli. Translation of the base sequence to the primary sequence revealed a transporter that is not homologous to any known secondary transporter. However, CitM shares 60% sequence identity with the gene product of open reading frame N15CR that is on the genome of B. subtilis and which has no known function. Open reading frame N15CR was cloned and expressed in E. coli and discovered to be a citrate transporter too.

The catalytic efficiency of 18 pig citrate synthase mutants were studied. The residues mutated were chosen according to the conservation of that residue in all known citrate synthase sequences, and the significance of that residue in substrate-amino acid interactions. Preliminary electrostatic calculations support the theory that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an 'enolized' version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible function for His274 is to stabilize this charge by delocalization mediated by a hydrogen bond.

The energetic effects of citrate metabolism in whole resting cells of Leuconostoc mesenteroides were studied. The generation of metabolic energy in the form of a pH gradient and a membrane potential by citrate metabolism was discovered to be primarily dependent on cometabolism with glucose. The rates of citrate use and of pyruvate and lactate production increased substantially when glucose is present, suggesting that glucose metabolism improves citrate metabolism.

The citrate permease (citP) gene appears to be plasmid encoded in Leuconostoc species and is highly homologous to the citP gene of Lactococcus lactis subsp. lactis biovar diacetylactis NCDO176. Citrate transport is required for the bacterial production of diacetyl and CO2 from citrate in cheese products. Homology with the L. lactis citP gene allowed the cloning of this gene from several Leuconostoc strains.

A study was conducted on isocitrate characterized from Cephalosporium acremonium CW-19 from cultures raised with poly(oxyethylene)sorbitan monopalmitate as the carbon source. The Mg2+-isocitrate complex was found to be the true substrate, while Mg2+ ions were found to function as anonessential activator. Results showed that Mg2+ ions produced total competitive protection. The presence of a histidine residue affecting the catalysis of the Phycomyces enzyme was suggested.

Analysis of the modulation of transcription of the citrate synthase genes citZ and citA in Bacillus subtilis reveals that after induction of the drug decoyinine into the bacterial cells, the citZ mRNA level was five times higher than the citA mRNA level. The citA gene transcription level is six times less with glucose as carbon source than with succinate as carbon source. Glutamate and glucose caused a 10-fold reduction and glucose alone causes a 2-fold reduction in the transcription of citZ gene. CitR protein from LysR family inhibits citA but not citZ gene expression.

Genetic complementation of the citrate synthase mutant strain W620 of Escherichia coli and PCR-based probing of the chromosomal DNA of Bacillus subtilis help isolate two different citrate synthase genes citZ and citA from B. subtilis. Null mutations in the citA gene have no influence on sporulation or citrate synthase enzyme functions, while citZ gene mutations induce severe abnormalities in sporulation and reduce citrate synthase enzyme functions. The citA gene was present in a monocistronic transcriptional unit while the citZ gene was present in a tricistronic cluster, coexisting with citH and citC genes.

Escherichia coli's gltA gene encodes citrate synthase, an enzyme of the tricarboxylic acid cycle pathway. In vivo examination of the expression of a gltA-lacZ fusion in response to anaerobiosis, different carbon substrates and changes in medium richness help study the regulation of gltA gene in E. coli. Aerobic conditions increase the expression by two-fold in comparison with that of anaerobic conditions. Delta-arcA function suppresses gltA expression during both conditions. Richness of the medium changes the expression of gltA-lacZ by 10 to 14 times during aerobic and anaerobic growth. Expression of gltA-lacZ exhibits an inverse relationship with cell growth rate, and regulation of gltA expression varies under different cell culture conditions.

Studies conducted on the kinetics and genetics of citrate transport in Leuconostoc oenos show that the metabolism and citrate uptake cause the formation of an electrochemical gradient of protons across the cytoplasmic membrane. The citrate transport is an electrogenic process where a uniport mechanism assists the translocation of H2cit-. Energy generation during citrate utilization occurs due to the uptake of citrate anions, which yield a membrane potential and form H+-consuming reactions that yield a pH gradient.

Uranyl citrate complex was exposed to visible light to induce its photodegradation in anaerobic and aerobic environments. The experiment was carried out to investigate a new photodegradation-biodegradation remediation scheme which allows the recovery of uranium. It also showed the influence of oxygen and pH level on the rate and extent of photodegradation, uranium speciation and the amount of intermediate degradation products.

Studies of sodium citrate-water buffers containing d-limonene reveal that d-limonene is oxidated less in samples containing low density polyethylene. The sorption into polymer prevents oxidation beyond the levels seen in the initial storage conditions. This sorption process may enhance the flavor of commercial orange juice by removing limonene that result in undesirable off-flavors.

Intense exercise may negatively influence the optimal glycolytic flux in gastrocnemius, and the influence is more prominent in 24-month-old group of female Fischer rats. There is no variation in the activities of citrate synthase, phosphofructokinase, 3-hydroxyacyl-CoA dehydrogenase and succinate dehydrogenase, with intense exercise in the skeletal muscle fiber type soleus. There is a significant reduction in the PFK activity in the muscle fibers, gastroenemius and red gastrocnemius as a result of exercise.

Intrinsic viscosities of Tamm-Horsfall glycoprotein (THP) at 200 mM NaCl and pH 5.7 and influence of THP on calcium oxalate monohydrate (COM) crystal aggregation (AC) in vitro are determined to examine the influence of citrate and calcium on these parameters. Promotive THP is transformed into restrictors by citrate at 3.5 mM. Recurrent calcium stone promoters facilitate the formation of COM crystal.

Acetyl-CoA enol acts as an intermediate in the citrate synthase reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA and His274 acting as an acid, donating a proton to the carbonyl. CS-oxaloacetate complexes with the transition-state analog inhibitor, carboxymethyl-CoA, mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. In comparison with the value of the chemical shift of the protonated carboxymethyl-CoA carboxyl in acidic aqueous solution or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low.

Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA, are similar to those with the activated form of acetyl-CoA. The X-ray structure of the ternary complex of the enzyme, oxaloacetate and carboxymethyl-CoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form.

A study was conducted to resolve the crystal structures of mitochondrial aconitase with isocitrate and nitroisocitrate bound. A bovine heart enzyme was used to determine the structure with nitroisocitrate bound while a porcine heart enzyme was used to determine the structire with isocitrate bound. A minimum of 23 residues sourced from all four domains of aconitase were found to contribute to the active site.

Aluminum when in soluble form, as found in acidic soils that comprise about 40 percent of the world's arable land, is toxic to many crops. Organic acid excretion has been correlated with aluminum tolerance in higher plants. Overproduction of citrate was shown to result in aluminum tolerance in transgenic, tobacco (Nicotiana tabacum) and papaya (Carica papaya) plants., When solubilized in acid soils, Al (primarily in the form of [Al.sup.3+]) is toxic to many crops and is the major limiting factor for plant productivity in these soils. Soil [...]

A.G. Oxford's breakthrough on the role of citrate in the carboxylic acid cycle was a common incident in which a simple solution to the problem was overlooked by more experienced biochemists. Hans Krebs erroneously proposed that citrate undergoes successive reactions to produce oxaloacetate and two molecules of carbon. The role of citrate in the citric acid cycle baffled biochemists until Oxford provided a solution based on the reactivity of the two -CH2COOH units of the asymmetric enzyme. Furthermore, the solution was so simple that it was overlooked by renowned biochemists.

Involvement of tyrosine kinase in citrate-stimulated aldosterone production in bovine glomerulosa cells. Am J Physiol Endocrinol Metab 279: E140-E145, 2000.--The present study was designed to assess whether citrate stimulates aldosterone production by isolated bovine adrenal glomerulosa cells in vitro. When the cells were incubated with graded concentrations of citrate up to 4.0 mM, basal aldosterone production was significantly elevated, with a gradual reduction of extracellular ionized calcium concentration. Without citrate, however, adding increasing amounts of calcium chloride to a calcium-free medium did not reproduce the citrate's effect on basal aldosterone production. Genistein, an inhibitor of tyrosine kinases, inhibited the citrate (4 mM)-induced aldosterone production in a dose-dependent manner, with 89.8% of inhibition at a concentration of 10 [micro]M. When the cells were exposed to citrate (4 mM) for 5, 10, and 30 min, tyrosine in Mr 105,000 endogenous protein was dominantly phosphorylated. This study demonstrates for the first time that citrate stimulates aldosterone production in bovine adrenal glomerulosa cells in vitro and also suggests a crucial involvement of protein tyrosine kinase in the steroidogenic action of citrate in the cells. tyrosine phosphorylation; membrane potential; genistein; steroidogenesis

Hering-Smith, Kathleen S., Cecilia T. Gambala, and L. Lee Hamm. Citrate and succinate transport in proximal tubule cells. Am. J. Physiol. Renal Physiol. 278: F492-F498, 2000.--Urinary citrate, which inhibits calcium nephrolithiasis, is determined by proximal reabsorption via an apical dicarboxylate transporter. Citrate is predominantly trivalent at physiological pH, but [citrate.sup.-2] is transported at the apical membrane. We now demonstrate that low-Ca solutions induce transport of [citrate.sup.-2] and succinate in opossum kidney cells. With 1.2 mM extracellular Ca, citrate uptake was pH insensitive and not competed by [succinate.sup.2]. In contrast, with low extracellular Ca, citrate uptake increased twofold, was inhibited by succinate (and other dicarboxylates), was stimulated by lowering extracellular pH (consistent with [citrate.sup.-2] transport), and increased further by lowering extracellular Mg. The effect of Ca was incrementally concentration dependent, between 0 and 1.2 mM. The effect of Ca was not simply complexation with citrate because succinate (which is complexed significantly less) was affected by Ca similarly. Incubation of cells for 48 h in a low-pH media increased citrate transport (studied at control pH) more than twofold, suggesting induction of transporters. dicarboxylate; calcium; magnesium; acid-base; nephrolithiasis

The synthesis, isolation, structural characterization and solution behavior of the first mononuclear aluminum citrate complex illustrate the significance of the interaction between aluminum and citrate in biological media. The chemical and structural characteristics of the low molecular weight aluminum citrate species help provide insight into citrate's ability to enhance aluminum's absorption and thus impact on its accumulation and biotoxicity at biological sites.

The aqueous coordination chemistry of iron with citric acid was investigated. Specifically, the synthesis, spectroscopic and structural properties of the first mononuclear, water soluble iron-citrate complex, (NH4)5Fe(C6H4O7)2*2H2O, were described. Reaction between iron(III) nitrate and citric acid with a 1:2 molar ratio in aqueous solution resulted in the isolation of yellow crystalline material consistent with the molecular formula (NH4)5Fe(C6H4O7)2*2H2O.

X-ray crystallographic studies help determine the structures of two synthesized dinuclear ferric citrate complexes. One of the compounds has two octahedrally coordinated alkoxide-bridged iron atoms with a common edge. The second compound has three bridging citrate ligands and each ligand employs its alkoxide group to bind two ferric ions. Magnetic susceptibility studies reveal the second compound to be a ferromagnetically coupled face-sharing d5-d5 system.

A study was conducted to analyze the functions of catalytic residues and the characteristics of the nucleophilic intermediate in citrate synthase. Computations were performed on models of the first stage of the reaction with ab initio and semiempirical techniques. Results suggested that the hydrogen bond between His-274 and the intermediate was not the low-barrier type. Findings also showed that the stabilization of the enolate supported by the hydrogen bond with the imidazole is affected by the interaction with the third component of the reacting system.