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PubMed1990

Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells.

1. Maximal activities of some key enzymes of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and glutaminolysis were measured in homogenates from a variety of normal, neoplastic and suppressed cells. 2. The relative activities of hexokinase and 6-phosphofructokinase suggest that, particularly in neoplastic cells, in which the capacity for glucose transport is high, hexokinase could approach saturation in respect to intracellular glucose; consequently, hexokinase and phosphofructokinase could play an important role in the regulation of glycolytic flux in these cells. 3. The activity of pyruvate kinase is considerably higher in tumorigenic cells than in non-tumorigenic cells and higher in metastatic cells than in tumorigenic cells: for non-tumorigenic cells the activities range from 28.4 to 574, for tumorigenic cells from 899 to 1280, and for metastatic cells from 1590 to 1627 nmol/min per mg of protein. 4. The ratio of pyruvate kinase activity to 2 x phosphofructokinase activity is very high in neoplastic cells. The mean is 22.4 for neoplastic cells, whereas for muscle from 60 different animals it is only 3.8. 5. Both citrate synthase and isocitrate dehydrogenase activities are present in non-neoplastic and neoplastic cells, suggesting that the full complement of tricarboxylic-acid-cycle enzymes are present in these latter cells. 6. In neoplastic cells, the activity of glutaminase is similar to or greater than that of hexokinase, which suggests that glutamine may be as important as glucose for energy generation in these cells.

Cell Line↗
PubMed1999

Coordinate induction of energy gene expression in tissues of mitochondrial disease patients.

We have examined the transcript levels of a variety of oxidative phosphorylation (OXPHOS) and associated bioenergetic genes in tissues of a patient carrying the myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) A3243G mitochondrial DNA (mtDNA) mutation and the skeletal muscles of 14 patients harboring other pathogenic mtDNA mutations. The patients' tissues, which harbored 88% or more mutant mtDNA, had increased levels of mtDNA transcripts, increased nuclear OXPHOS gene transcripts including the ATP synthase beta subunit and the heart-muscle isoform of the adenine nucleotide translocator, and increased ancillary gene transcripts including muscle mitochondrial creatine phosphokinase, muscle glycogen phosphorylase, hexokinase I, muscle phosphofructokinase, the E1alpha subunit of pyruvate dehydrogenase, and the ubiquinone oxidoreductase. A similar coordinate induction of bioenergetic genes was observed in the muscle biopsies of severe pathologic mtDNA mutations. The more significant coordinated expression was found in muscle from patients with the MELAS, myoclonic epilepsy with ragged red fibers, and chronic progressive external ophthalmoplegia deletion syndromes, with ragged red muscle fibers and mitochondrial paracrystalline inclusions. High levels of mutant mtDNAs were linked to a high induction of the mtDNA and nuclear OXPHOS genes and of several associated bioenergetic genes. These observations suggest that human tissues attempt to compensate for OXPHOS defects associated with mtDNA mutations by stimulating mitochondrial biogenesis, possibly mediated through redox-sensitive transcription factors.

Adolescent↗
PubMed1976

Acetyl-CoA production and utilization during growth of the facultative methylotroph Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate.

In Pseudomonas AM1, conversion of 3-hydroxybutyrate to acetyl-CoA is mediated by an inducible 3-hydroxybutyrate dehydrogenase, an acetoacetate: succinate coenzyme A transferase (specific for succinyl-CoA) and an inducible beta-ketothiolase. Ethanol is oxidized to acetate by the same enzymes as are involved in methanol oxidation to formate. An inducible acetyl-CoA synthetase has been partially purified and characterized; it is essential for growth only on ethanol, malonate and acetate plus glyoxylate, as shown by the growth characteristics of a mutant (ICT54) lacking this enzyme. Free acetate is not involved in the assimilation of acetyl-CoA, and hydroxypyruvate reductase is not involved in the oxidation of acetyl-CoA to glyoxylate during growth on 3-hydroxybutyrate. A mutant (ICT51), lacking 'malate synthase' activity has been isolated and its characteristics indicate that this activity is normally essential for growth, of Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate, but not for growth on other substrates such as pyruvate, succinate and C1 compounds. The growth properties of a revertant (ICT51R) and of a mutant lacking malyl-CoA lyase (PCT57) indicate that an alternative route must exist for assimilation of compounds metabolized exclusively by way of acetyl-CoA.

Acetate-CoA Ligase↗
PubMed1986

Metabolic changes associated with adaptation of plant cells to water stress.

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from (15)N labeling kinetics after substituting [(15)N]ammonium and [(15)N]nitrate for the (14)N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and gamma-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and gamma-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and gamma-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low (15)N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.

Journal Article↗
PubMed1983

Control of the activity of phosphoenolpyruvate carboxykinase and the level of its mRNA in livers of newborn rats. Effect of diabetes, glucose load and glucocorticoids.

Streptozotocin treatment produces a typical experimental diabetes in neonates exhibiting hyperglycemia, glucosuria, ketonemia and increased level of fatty acids in the blood. The liver is affected as well, with reduced activity of glycogen synthase and a corresponding decrease in the content of liver glycogen. In contrast, the activity of liver cytosolic phosphoenolpyruvate carboxykinase and the level of its mRNA are not affected. Using a cDNA containing P-pyruvate carboxykinase sequence, the relative abundance of the enzyme mRNA was estimated. The level of the mRNA was readily observed increasing by glucocorticoid treatment or decreasing in response to administered load of glucose. These parallel the changes observed in the activity of the enzyme under these treatments, indicating that the level of P-pyruvate carboxykinase mRNA actually determines that of the enzyme. The failure of diabetes to increase the level of enzyme mRNA and the limited response to glucose loading strongly suggest that the mechanisms controlling the level of P-pyruvate carboxykinase mRNA in neonates are relatively resistant to insulin. This is unique to neonates, since in both the adult and the fetal liver. P-pyruvate carboxykinase readily responds to insulin. The minimal levels of glucocorticoids characteristic of neonates may be associated with this phenomenon.

Animals↗
PubMed2000

Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects.

Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum-plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (alpha- and beta-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P(II). The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (gamma-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a P(II)-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O(2) level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.

Anthranilate Synthase↗
PubMedDate not supplied

[Metabolism of C2-C6-substrates under mixotrophic growth of Acinetobacter sp. B-7005 and B-7005 (1HG) strains].

Activities of the key enzymes of C2-C6-metabolism were assayed under cultivation of Acinetobacter sp. B-7005 and B-7005 (1Hgamma) strains on ethanol and glucose mixture. Under mixotrophic growth of bacteria the enzymes activity of ethanol metabolism (NAD+ -dependent alcohol dehydrogenase, NADP+ -dependent acetaldehyde dehydrogenase, acetyl-KoA-synthetase) and glucose metabolism (6-phosphofructokinase and 6-phosphogluconate dehydratase) was lower than that on corresponding monosubstrates. The activity of isocitrate lyase and malate synthase in cells grown on the substrate mixture declined to an even greater extent, indicating that the role of the glyoxylate cycle in such cells is insignificant. The simultaneous functioning of the glyoxylate cycle and pyruvate carboxylase reaction, increasing of phosphoenolpyruvate synthetase activity testify to the gluconeogenesis intensification under mixotrophic growth of Acinetobacter sp. B-7005 and B-7005 (1Hgamma).

Acinetobacter↗
PubMed1983

Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3 -methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) converts HMG-CoA to mevalonate, a key intermediate in the formation of cholesterol and several nonsterol isoprenoid compounds. Using the bromodeoxyuridine/bisbenzimide photosensitization technique, we isolated a mutant clone of Chinese hamster ovary cells that requires mevalonate for growth. This mutant, designated UT-2 cells, expresses 2-5% of the HMG-CoA reductase activity of parental Chinese hamster ovary cells, even after growth for 9 months in the absence of selective pressure. By immunoblotting, no cross-reactive HMG-CoA reductase protein was detected in UT-2 cells. Incorporation of [14C]acetate and [14C]pyruvate into cholesterol was less than 5% of that in parental Chinese hamster ovary cells. In contrast, [3H]mevalonate incorporation into cholesterol was normal. The activities of acetoacetyl-CoA thiolase and HMG-CoA synthase, the two enzymes that precede HMG-CoA reductase in the cholesterol biosynthetic pathway, were normal or slightly elevated in UT-2 cells. No gross deletions or rearrangements in the gene for HMG-CoA reductase were apparent when DNA from UT-2 cells was digested with restriction endonucleases, subjected to Southern blotting, and probed with a 32P-labeled cDNA for HMG-CoA reductase. We conclude that UT-2 cells have a mutation that specifically prevents the production of normal amounts of HMG-CoA reductase.

Animals↗
PubMed1989

Formation of ketone bodies by resting lymphocytes.

1. Both beta-hydroxy-beta-methylglutaryl-coenzyme A synthase and lyase activities are present in rat mesenteric lymphocytes: all of the synthase and almost all (80%) of the lyase were present in the mitochondrial compartment of the cell. 2. A high rate of acetoacetate formation was observed in mesenteric lymphocytes incubated in vitro for 60 min in the absence of added substrate; addition of pyruvate or glutamine increased the "endogenous" rate of acetoacetate formation by about 30%. 3. The rates of ketone body formation are similar to maximal rates observed for rat liver. 4. It is suggested that the high rate of endogenous acetoacetate production occurs from long chain fatty acids: this suggestion is consistent with the reported high "endogenous" rate of O2 consumption by lymphocytes. 5. Of the pyruvate metabolized via pyruvate dehydrogenase in lymphocytes, ca 50-70% could be accounted for as acetoacetate, acetate, 3-hydroxybutyrate and citrate: the fate of the remainder is not known. 6. There was a high rate of endogenous acetoacetate formation by isolated mitochondria from these cells. 7. The rate was doubled by addition of pyruvate or butyrate; it was trebled by addition of propionate, ADP or carbonyl cyanide trichloro-methoxyphenylhydrazone; but it was decreased by addition of antimycin A or glutamine. 8. It is suggested that the high rates of acetoacetate formation in these cells acts as a dynamic "buffer" system for the acetyl coenzyme A (CoA) concentration: that is, acetyl CoA is always available for fatty acid synthesis, cholesterologenesis, chain extension of fatty acids or acetylation of proteins (e.g. for covalent control of their activity) which will be demanded at different stages of the cell cycle. 9. This is another example of branch-point sensitivity in control in cells with the potential for rapid cell division.

Acetoacetates↗
PubMed1999

Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides.

The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant. The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type gltA with the gltA::kan allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0. 2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.

Chromatography, High Pressure Liquid↗
PubMed1988

Studies on the biosynthesis of bialaphos (SF-1293). 8. Purification and characterization of 2-phosphinomethylmalic acid synthase from Streptomyces hygroscopicus SF-1293.

2-Phosphinomethylmalic acid (PMM) synthase catalyzes the condensation of phosphinopyruvic acid (PPA), an analog of oxalacetic acid, and acetyl-CoA to form PMM. The enzyme was purified approximately 700-fold from a cell-free extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism, to an electrophoretically homogeneous state. The purified PMM synthase has a subunit molecular weight of 48,000 by SDS-polyacrylamide gel electrophoresis and a native molecular weight of 90,000 approximately 98,000 by gel filtration. PMM synthase was relatively unstable, showed maximum activity at pH 8.0 and 30 degrees C, and was inhibited strongly by p-chloromercuribenzoate, iodoacetamide and EDTA. Enzyme activity suppressed by EDTA was completely restored by adding Co++ or Mn++ and partially restored by addition of Ca++, Fe++ or Mg++. The specific substrates of this enzyme are PPA or oxalacetic acid in addition to acetyl-CoA. The enzyme does not catalyze the liberation of CoA from acetyl-CoA in the presence of alpha-keto acids, such as pyruvate, alpha-ketoglutarate, deamino-alpha-ketodemethylphosphinothricin or phosphonopyruvate. The condensation reaction did not take place when propionyl-CoA or butyryl-CoA was used as a substrate in place of acetyl-CoA. The Km values of the enzyme were 0.05 mM for acetyl-CoA, 0.39 mM for PPA and 0.13 mM for oxalacetate. PMM synthase is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity; unlike (R)-citrate synthase PMM synthase was not inhibited by oxygen.

Chromatography, Gel↗
PubMed2006

Mitochondrial fatty acid synthesis in Trypanosoma brucei.

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radio-labeled fatty acids in cultured procyclic parasites with [(14)C]pyruvate or [(14)C]threonine, either of which is catabolized to [(14)C]acetyl-CoA in the mitochondrion. Although some of the [(14)C]acetyl-CoA may be utilized by the ELO pathway, a striking reduction in radiolabeled fatty acids following ACP RNAi confirmed that it is also consumed by mitochondrial FAS. ACP depletion by RNAi or gene knockout also reduces lipoic acid levels and drastically decreases protein lipoylation. Thus, octanoate (C8), the precursor for lipoic acid synthesis, must also be a product of mitochondrial FAS. Trypanosomes employ two FAS systems: the unconventional ELO pathway that synthesizes bulk fatty acids and a mitochondrial pathway that synthesizes specialized fatty acids that are likely utilized intramitochondrially.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1984

Malyl-CoA formation in the NAD-, CoASH-, and alpha-ketoglutarate dehydrogenase-dependent oxidation of 2-keto-4-hydroxyglutarate. Possible coupled role of this reaction with 2-keto-4-hydroxyglutarate aldolase activity in a pyruvate-catalyzed cyclic oxidation of glyoxylate.

The alpha-ketoglutarate dehydrogenase complex of either pig heart or Escherichia coli catalyzes a NAD- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate which is stereoselective toward the L-isomer of this hydroxyketo acid. L-Malyl-CoA is the product of the reaction; the evidence includes observing (a) a steady increase in absorbance at 230 nm during the oxidation of 2-keto-4-hydroxyglutarate, (b) a positive response of oxidation reaction mixtures to neutral hydroxylamine, (c) loss of the two foregoing results concomitant with release of thiol-reacting material and the formation of free malate when reaction mixtures are heated, (d) formation of a hydroxamate which has chromatographic mobilities identical to that of chemically synthesized malate hydroxamate, (e) enzymatic formation of a radioactive product from 14C-labeled 2-keto-4-hydroxyglutarate which co-migrates with chemically synthesized malyl-CoA, and (f) hydrolysis of the product by citrate synthase, an enzyme absolutely specific for citryl-CoA and L-malyl-CoA. A 1:1:1 stoichiometric relationship exists between the amount of 2-keto-4-hydroxyglutarate oxidized, NAD reduced, and malate (or malyl-CoA) formed. Results from studies in which either 14C-labeled 2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate is incubated with mixtures of purified enzymes or extracts of E. coli support the suggestion that the aldolase which preferentially catalyzes formation of L-2-keto-4-hydroxyglutarate from pyruvate plus glyoxylate in E. coli is coupled with the oxidative decarboxylation of this substrate, as reported here, and other enzymes in a multistep pyruvate-catalyzed cyclic oxidation of glyoxylate.

Acyl Coenzyme A↗
PubMed1994

Insulin-like actions of tungstate in diabetic rats. Normalization of hepatic glucose metabolism.

Oral administration of tungstate for 15 days normalized glycemia in streptozotocin-induced diabetic rats. Simultaneously, the alterations in hepatic glucose metabolism due to diabetes were almost completely counteracted by this treatment. Thus, 6-phosphofructo-2-kinase, L-pyruvate kinase, and glycogen phosphorylase alpha activities reached levels similar to those observed in healthy animals. Hepatic levels of fructose 2,6-bisphosphate and glycogen also recovered. However, the recovery of glucokinase activity and hepatic levels of glucose 6-phosphate was only partial. The total activity of glycogen synthase increased, although the activation state was not recovered. Moreover, mRNA levels of hepatic glucokinase, glycogen phosphorylase, and phosphoenolpyruvate carboxykinase were also normalized. Tungstate administration in healthy animals also affected all these parameters, although to a much lesser extent. All these effects were similar to those previously reported for vanadate, suggesting a common mechanism of action in vivo.

Animals↗
PubMed2004

Antioxidants inhibit angiogenesis in vivo through down-regulation of nitric oxide synthase expression and activity.

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H2O2) scavenger sodium pyruvate decreased, while H2O2 increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and L-NAME, but not D-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.

Allantois↗
PubMed1999

Metabolite and isotopomer balancing in the analysis of metabolic cycles: II. Applications.

In a previous paper (Klapa et al., 1999), we presented a model for the analysis of isotopomer distributions of the TCA cycle intermediates resulting from 13C (or 14C) labeling experiments. Results allow the rigorous determination of the degree of enrichment at specific carbon atoms of metabolites, of the molecular weight distribution of metabolite isotopomers, as well as of the fine structure of NMR spectra in terms of a small number of metabolic fluxes. In this paper we validate the model by comparing model predictions with experimental data and then apply it to the analysis of metabolic networks that have been investigated in previous studies. The results have allowed us to conclude that: (1) there is no evidence of propionyl-CoA carboxylase pathway in Escherichia coli; and (2) the possibility that acetone utilization in mammals occurs solely via the "lactate/methylglyoxal" pathway is consistent with available labeling data. The presented modeling framework provides additional constraints that must be satisfied by experimental data in a biochemical network structure and therefore enhances the power of labeling methods for resolving in vivo metabolic fluxes.

Acetic Acid↗