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Byline: S. Z. H. Shah, M. Fatima, M. Afzal, S. M. Hussain, H. Azmat, M. Bilal and T. Ahmed Key words: Phytase, citric acid, rohu(Labeo rohita), bone mineralization, body mineralization, [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.04.031 Byline: Xin Zhou (a)(b), Kathryn V. Anderson (a) Keywords: Mouse embryo; Forebrain specification; Morphogenesis; Mitochondrial metabolism; Lipoic acid; Prechordal plate; Lipoic acid synthetase; AMPK; Cyclin E; Amish microcephaly; Glycolysis; Citric acid cycle; Microcephaly Abstract: Mouse genetic studies have defined a set of signaling molecules and transcription factors that are necessary to induce the forebrain. Here we describe an ENU-induced mouse mutation, nearly headless (nehe), that was identified based on the specific absence of most of the forebrain at midgestation. Positional cloning and genetic analysis show that, unlike other mouse mutants that disrupt specification of the forebrain, the nehe mutation disrupts mitochondrial metabolism. nehe is a hypomorphic allele of Lipoic acid Synthetase (Lias), the enzyme that catalyzes the synthesis of lipoic acid, an essential cofactor for several mitochondrial multienzyme complexes required for oxidative metabolism. The defect in forebrain development in nehe mutants is apparent as soon as the forebrain is specified, without a concomitant increase in apoptosis. Two tissues required for forebrain specification, the anterior visceral endoderm and the anterior definitive endoderm, develop normally in nehe mutants. However, a third head organizer tissue, the prechordal plate, fails to express markers of cell type determination and shows abnormal morphology in the mutants. We find that the level of phosphorylated (active) AMPK, a cellular energy sensor that affects cell polarity, is up-regulated in nehe mutants at the time when the prechordal plate is normally specified. The results suggest that the nehe phenotype arises because high levels of energy production are required for the specialized morphogenetic movements that generate the prechordal plate, which is required for normal development of the mammalian forebrain. We suggest that a requirement for high levels of ATP for early forebrain patterning may contribute to certain human microcephaly syndromes. Author Affiliation: (a) Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10065, USA (b) Biochemistry & Structural Biology, Cell and Developmental Biology, and Molecular Biology Program, Weill Graduate School of Medical Sciences, Cornell University, 1300 York Avenue, New York, NY 10065, USA Article History: Received 27 January 2010; Revised 26 April 2010; Accepted 27 April 2010

Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coil citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant ~2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coil gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525. Significantly low extracellular citrate levels as compared to the intracellular levels in Pf(pAB7) suggested a probable limitation of efficient citrate transport.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.08.040 Byline: Jean B. Regard (1), Isaac T. Sato (1), Shaun R. Coughlin (1) Keywords: RNA; MOLNEURO; SIGNALING Abstract: G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease processes. To better understand the functions of GPCRs in vivo, we quantified transcript levels of 353 nonodorant GPCRs in 41 adult mouse tissues. Cluster analysis placed many GPCRs into anticipated anatomical and functional groups and predicted previously unidentified roles for less-studied receptors. From one such prediction, we showed that the Gpr91 ligand succinate can regulate lipolysis in white adipose tissue, suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis. We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects. This resource will aid studies to understand GPCR function in vivo and may assist in the identification of therapeutic targets. Author Affiliation: (1) Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA Article History: Received 29 January 2008; Revised 27 June 2008; Accepted 28 August 2008 Article Note: (miscellaneous) Published: October 30, 2008

In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, L-alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA. Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

Byline: Luca Gabutti (1), Claudio Marone (3), Giuseppe Colucci (1), Francesca Duchini (1), Carlo Schonholzer (2) Keywords: Continuous renal replacement therapy Continuous venovenous hemodiafiltration Regional citrate anticoagulation Abstract: Abstract Objective. Feasibility and safety evaluation of regional citrate anticoagulation (RCA) versus systemic heparinization for continuous venovenous hemodiafiltration. Design and setting. Combined retrospective and prospective observational study performed in a secondary multidisciplinary intensive care unit of the Ospedale Civico Lugano Switzerland. Patients and interventions. Twelve hemodynamically unstable patients (median APACHE II score 26, interquartile range 22--29) in whom heparin was judged to be at least temporarily contraindicated. A switch from RCA (predilution setting same iso-osmotic replacement and dialysis fluid) to heparinization or vice versa was recommended for the final evaluation 56 dialyzers were used for RCA (1400 h) and 39 for heparinization (1271 h). Measurements and results. Median dialyzer life span was 24.2 h (interquartile range 17.4--42.3) for RCA and 42.5 h (20.6--69.1) for heparinization. Fluid control and dialysis quality were similar in the two groups and required no additional intervention. The risk of significant hypocalcemia and metabolic alkalosis was higher at the beginning of the RCA program and decreased with the further training of the staff. Seven bleeding episodes occurred with heparinization vs. three in RCA. Conclusions. RCA may be a safe and useful form of anticoagulation which is more expensive than heparinization but helps to minimize bleeding risk. The risk of metabolic complications is higher at the beginning of a new RCA program. For centers lacking experienced staff we suggest reserving this technique for patients with rapid clotting of the extracorporeal circuit if treated without anticoagulation. Author Affiliation: (1) Department of Internal Medicine, Ospedale la Carita Locarno, 6600 Locarno, Switzerland (2) Department of Nephrology, Ospedale Civico Lugano, Switzerland (3) Department of Internal Medicine, Ospedale San Giovanni Bellinzona, Switzerland Article History: Received Date: 25/03/2002 Accepted Date: 16/07/2002 Article note: Electronic Publication

Little is known about the sources of cytosolic acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of fatty acid oxidation in the heart. We tested the hypothesis that citrate provides acetyl-CoA for malonyl-CoA synthesis after its mitochondrial effiux and cleavage by cytosolic ATP-citrate lyase. We expanded on a previous study where we characterized citrate release from perfused rat hearts (Vincent G, Comte B, Poirier M, and Des Rosiers C. Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846-E856, 2000). In the present study, we show that citrate release rates, ranging from 6 to 22 nmol/min, can support a net increase in malonyl-CoA concentrations induced by changes in substrate supply, at most 0.7 nmol/min. In experiments with [U-[sup.13]C](lactate + pyruvate) and [1-[sup.13]C]oleate, we show that the acetyl moiety of malonyl-CoA is derived from both pyruvate and long-chain fatty acids. This [sup.13]C-labeling of malonyl-CoA occurred without any changes in its concentration. Hydroxycitrate, an inhibitor of ATP-citrate lyase, prevents increases in malonyl-CoA concentrations and decreases its labeling from [U-[sup.13]C](lactate + pyruvate). Our data support at least a partial role of citrate in the transfer from the mitochondria to cytosol of acetyl units for malonyl-CoA synthesis. In addition, they provide a dynamic picture of malonyl-CoA metabolism: even when the malonyl-CoA concentration remains constant, there appears to be a constant need to supply acetyl-CoA from various carbon sources, both carbohydrates and lipids, for malonyl-CoA synthesis. gas chromatography-mass spectrometry; adenosine 5'-triphosphate-citrate lyase; hydroxycitrate; acetyl-coenzyme A; citric acid cycle; [sup.13]C-substrate; isotopomer analysis

We examined the extent to which maintenance diet influences the taste preferences of mice. C57BL/6J (B6) and 129X1/SvJ (129) mice were fed one of three standard cereal-based diets (Teklad 8604, Zeigler NIH-07, Purina 5001), a cereal-based diet formulated for breeding (Purina 5015), or two purified diets (AIN-76A or AIN-93G). The mice were given 48-h two-bottle choice tests between water and the following seven taste solutions: 2 mmol/L saccharin, 5 mmol/L citric acid, 50 mmol/L citric acid, 30 [micro]mol/L quinine hydrochloride (QHCl), 300 [micro]mol/L QHCl, 75 mmol/L NaCl, and 10% ethanol. There were very few differences in taste solution preference scores among mice of the same strain fed the three different versions of standard cereal-based diet. There were also very few differences in taste solution preference scores between mice of the same strain fed the two purified diets. However, the mice fed standard cereal-based diets generally drank more water and total fluid than did mice fed purified diets. There were larger differences between the B6 and 129 strains in saccharin and ethanol preference scores with mice fed standard cereal-based diets than purified diets. Conversely, there were larger differences between the B6 and 129 strains in citric acid and NaCl preference scores with mice fed purified diets than standard cereal-based diets. These results show that maintenance diet composition can have strain-dependent effects on taste solution preference. They illustrate that attention must be paid to the effects of diet on phenotype in screens of mutagenized mice and other genetic studies. KEY WORDS: * saccharin * citric acid * quinine * NaCl * ethanol * taste preference * mice

Research has been conducted on the cytoplasmic malate dehydrogenase. The regulation of this dehydrogenase and of he membrane-associated dehydrogenases, succinate and NADH dehydrogenases has been investigated.

Research has been conducted on the oxidation of malate to oxaloacetate in Escherichia coli. The creation of the deletion malic enzyme gene mutants and the phosphoenolpyruvate synthase gene in investigating whether the malic enzyme can take over the conversion of malate to oxaloacetate in an mqo mutant is described.

Oxalic acid production by Aspergillus niger is discussed. An mutant which does not produce oxalate and for which data is presented, produces citric acid if at pH 5 with manganese present. Activity of oxaloacetate acetylhydrolase (OAH), the enzyme thought to be responsible for oxalate formation in A. niger, correlated with oxalate production and external pH seemed to be the primary factor regulating oxalic acid production by A. niger.

Intrarenal control mechanisms play an important role in the maintenance of body fluid and electrolyte balance and pH homeostasis. Recent discoveries of new ion transport and regulatory pathways in the [...]

The effect of glycerol in mitochondrial citrate oxidation by Aspergillus niger was investigated. Mycelia of A. niger incubated with 0.5 molar glycerol increased intracellular glycerol pool level to 0.92 molar but had no effect on citrate uptake or carbon dioxide (CO2) release by the mycelia. Mitochondria preloaded with glycerol showed no significant increase in rate of CO2 release from mitochondria not loaded with glycerol. Loading with glycerol also had no effect on NADP-specific isocitrate dehydrogenase activity. These results suggest that glycerol neither stimulates nor inhibits citric acid accumulation by A. niger.

A new model is proposed for the estimation of the rate of the flux through the citric acid cycle (CAC) in heart without the use of differential equations. The conventional models require elaborate numerical solutions for differential equations which describe the CAC in the heart. The new model is accurate and also simple in design and usage.

A study of Methylophilus methylotrophus strain AS1 is studied with emphasis on the citric acid cycle and the pathway of 5-aminolevulinic acid to reveal the presence of isocitrate dehydrogenase. This enzyme is unusual for the selection of NAD, rather than NADP, as a coenzyme for the conversion of pyruvate to 2-oxoglutarate. The absence of the enzymes of the glyoxylate cycle indicate the exclusively biosynthetic role of the cycle.

Factors influencing the Aspergillus system were investigated in relation to the Penicillium system. The transport of citric acid across the plasma membrane of Penicillium simplicissimum and the influence of metabolic energy on the activity of vital enzymes of fungal plasma membrane were also examined. The H(+)-ATPase of Saccharomyces cerevisiae can be induced by extracellular buffer.

The Klebsiella pneumoniae CitS system is a Na+ -dependent citrate transport system which functions under anaerobic conditions. An investigation was conducted to determine the mechanism of citrate transport mediated by CitS. Analysis of the binding activities of different citrate species showed that preferred species is H-citrate(super 2-). Analysis of the stoichiometry of citrate transport indicates that every citrate molecule is transported via symport with one sodium and at least two hydrogen ions.

Factors contributing to phosphate solubilization were examined in Penicillium bilaii Chalabuda. Both the nutrient composition and buffering capacity of the medium affected the production of acidic metabolites which contribute to increased solubility. Citrate and oxalate were the major metabolites produced. Citrate was produced under nitrogen-limiting conditions, while oxalate was produced when carbon was limiting. Futhermore, citrate was produced in both log and stationary phases, while oxalate was produced only in the stationary phase. Sporulating cultures tended to produce less acid.

Post weanling pigs were given diets supplemented with either fumaric or citric acid to determine the effect of the supplements on intestinal acidity, chloride ion (Cl-) levels, volatile fatty acid content and microfloral profile. Weanlings were sacrificed and the content of the gastrointestinal tract was analyzed. Results show that organic acid supplements had no effect on intestinal acidity and Cl- concentrations. Likewise, volatile fatty acid content was not altered and no conclusive effects were observed on bacterial populations in the gastrointestinal tract.

The present experiment investigated Fos-like immunoreactivity (FLI) in the nucleus of the solitary tract (NST) after intraoral infusions of 0.1 M citric acid, 0.3 M NaCl, and 0.3-30 mM quinine monohydrochloride (QHCl) in awake, behaving rats. Increases in QHCl concentration produced increases in the numbers of FLI-labeled neurons in the rostral part of the intermediate ([i.sub.r]) and rostral (r) NST, but the topographic distribution of FLI was consistent across QHCl concentrations and distinctive compared with effects of citric acid. Quinine elicited FLI concentrated in the medial third of the nucleus; acid elicited more broadly distributed FLI concentrated farther laterally. Surprisingly, in contrast to QHCl and citric acid, NaCl produced FLI that was indistinguishable from that produced by water. Although the functional significance of these patterns is unknown, citric acid and QHCl are nonpreferred stimuli but produced different oromotor behaviors. QHCl (30 mM) elicited ~3.2 times as many gapes as citric acid (0.1 M), and acid elicited more ingestive responses. Parallel differences in FLI expression suggest that different NST regions may have distinctive roles in triggering oromotor behaviors. nucleus tractus solitarius; chemotopy; sodium chloride; parabrachial nucleus; gaping

Panchal, Ashish R., Blandine Comte, Hazel Huang, Todd Kerwin, Ahmed Darvish, Christine Des Rosiers, Henri Brunengraber, and William C. Stanley. Partitioning of pyruvate between oxidation and anaplerosis in swine hearts. Am J Physiol Heart Circ Physiol 279: H2390-H2398, 2000--The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts by infusing [[U-.sup.13][C.sub.3]]lactate and/or [[U-.sup.13][C.sub.3]] pyruvate into the left anterior descending (LAD) coronary artery. After 1 h, the tissue was freeze-clamped and analyzed by gas chromatography-mass spectrometry for the mass isotopomer distribution of citrate and its oxaloacetate moiety. LAD blood pyruvate and lactate enrichments and concentrations were constant after 15 min of infusion. Under near-normal physiological concentrations of lactate and pyruvate, pyruvate carboxylation and decarboxylation accounted for 4.7 [+ or -] 0.3 and 41.5 [+ or -] 2.0% of citrate formation, respectively. Similar relative fluxes were found when arterial pyruvate was raised from 0.2 to 1.1 mM. Addition of 1 mM octanoate to 1 mM pyruvate inhibited pyruvate decarboxylation by 93% without affecting carboxylation. The absence of M1 and M2 pyruvate demonstrated net irreversible pyruvate carboxylation. Under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the citric acid cycle flux despite considerable variation in the flux through pyruvate decarboxylation. cardiac; citric acid cycle; pyruvate dehydrogenase; pyruvate carboxylation; myocardial metabolism

The core of intermediary metabolism in autotrophs is the citric acid cycle. In a certain group of chemoautotrophs, the reductive citric acid cycle is an engine of synthesis, taking in [CO.sub.2] and synthesizing the molecules of the cycle. We have examined the chemistry of a model system of C, H, and O that starts with carbon dioxide and reductants and uses redox couples as the energy source. To inquire into the reaction networks that might emerge, we start with the largest available database of organic molecules, Beilstein on-line, and prune by a set of physical and chemical constraints applicable to the model system. From the 3.5 million entries in Beilstein we emerge with 153 molecules that contain all 11 members of the reductive citric acid cycle. A small number of selection rules generates a very constrained subset, suggesting that this is the type of reaction model that will prove useful in the study of biogenesis. The model indicates that the metabolism shown in the universal chart of pathways may be central to the origin of life, is emergent from organic chemistry, and may be unique.

Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846-E856, 2000.--Cytosolic citrate is proposed to play a crucial role in substrate fuel selection in the heart. However, little is known about factors regulating the transfer of citrate from the mitochondria, where it is synthesized, to the cytosol. Further to our observation that rat hearts perfused under normoxia release citrate whose [sup.13]C labeling pattern reflects that of mitochondrial citrate (B. Comte, G. Vincent, B. Bouchard, and C. Des Rosiers. J. Biol. Chem. 272: 26117-26124, 1997), we report here data indicating that this citrate release is a specific process reflecting the mitochondrial efflux of citrate, a process referred to as cataplerosis. Indeed, measured rates of citrate release, which vary between 2 and 21 nmol/min, are modulated by the nature and concentration of exogenous substrates feeding acetyl-CoA (fatty acid) and oxaloacetate (lactate plus pyruvate) for the mitochondrial citrate synthase reaction. Such release rates that represent at most 2% of the citric acid cycle flux are in agreement with the activity of the mitochondrial tricarboxylate transporter whose participation is also substantiated by 1) parallel variations in citrate release rates and tissue levels of citrate plus malate, the antiporter, and 2) a lowering of the citrate release rate by 1,2,3-benzenetricarboxylic acid, a specific inhibitor of the transporter. Taken together, the results from the present study indicate that citrate cataplerosis is modulated by substrate supply, in agreement with the role of cytosolic citrate in fuel partitioning, and occurs, at least in part, through the mitochondrial tricarboxylate transporter. energy metabolism; citric acid cycle; [sup.13]C mass isotopomer analysis; mitochondrial tricarboxylic acid transporter

In isotopic experiments, the labeling pattern of glutamate opens a window on hepatic metabolism, particularly the citric acid cycle, gluconeogenesis and fatty acid oxidation. This is because glutamate is in isotopic equilibrium with ([Alpha]-ketoglutarate, whose labeling pattern is influenced by the following: 1) the contributions of glucose and fatty acids to acetyl-CoA, 2) the relative contributions of pyruvate carboxylase and pyruvate dehydrogenase to the entry of pyruvate carbon into the citric acid cycle, and 3) the rate of gluconeogenesis in relation to citric acid cycle activity. In humans and primates, hepatic glutamate can be sampled noninvasively via urinary phenylacetylglutamine, which is formed in liver from phenylacetate (a side product of phenylalanine catabolism) and glutamine (which equilibrates with liver glutamate and [Alpha]-ketoglutarate). The [sup.14]C- or [sup.13]C-labeling pattern of the glutamate moiety of phenylacetylglutamine can be measured by sequential degradations to [sup.14][CO.sub.2], gas chromatography-mass spectrometry or nuclear magnetic resonance (NMR). When phenylacetylglutamine is labeled from singly labeled [[sup.14]C]- or [[sup.13]C]substrates, relative metabolic rates can be computed from the labeling pattern using Landau's model. In diabetic patients infused with [3-[sup.13]C]pyruvate, the noninvasive sampling of hepatic glutamate via phenylacetylglutamine allows one to test the degree of liver insulinization via the (pyruvate carboxylase)/ (pyruvate dehydrogenase) activity ratio. This ratio regulates gluconeogenesis in part. Its measurement may allow the identification of patients who might benefit from the intraperitoneal administration of insulin, or from recently developed antidiabetic drugs. J. Nutr. 130: 991S-994S, 2000. KEY WORDS: * phenylacetylglutamine * gluconeogenesis * tricarboxylic acid cycle * isotopic methods * diabetes