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PubMed2000

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

Acetyl-CoA C-Acetyltransferase↗
PubMed2006

Peroxisome proliferator-activated receptor-alpha selective ligand reduces adiposity, improves insulin sensitivity and inhibits atherosclerosis in LDL receptor-deficient mice.

Fenofibrate, a selective (1)PPAR-alpha activator, is prescribed to treat human dyslipidemia. The aim of this study was to delineate the mechanism of fenofibrate-mediated reductions in adiposity, improvements in insulin sensitivity, and lowering of triglycerides (TG) and free fatty acids (FFA) and to investigate if these favorable changes are related to the inhibition of lipid deposition in the aorta. To test this hypothesis we used male LDLr deficient mice that exhibit the clinical features of metabolic syndrome X when fed a high fat high cholesterol (HF) diet. LDLr deficient mice fed HF diet and simultaneously treated with fenofibrate (100 mg/kg body weight) prevented development of obesity, lowered serum triglycerides and cholesterol, improved insulin sensitivity, and prevented accumulation of lipids in the aorta. Lowering of circulating lipids occurred via down-regulation of lipogenic genes, including fatty acid synthase, acetyl CoA carboxylase and diacyl glycerol acyl transferase-2, concomitant with decreased liver TG and cholesterol, and TG output rate. Fenofibrate also suppressed liver apoCIII mRNA levels and markedly increased lipoprotein lipase mRNA levels, known to enhance serum TG catabolism. In addition, fenofibrate profoundly reduced epididymal fat and mesenteric fat mass to the levels seen in lean mice. The reductions in body weight were associated with elevation of hepatic uncoupling protein 2 (UCP2) mRNA, a concomitant increase in the ketone body formation, and improved insulin sensitivity associated with tumor necrosis factor-alpha reductions and phosphoenol pyruvate carboxykinase down-regulation. These results demonstrate that fenofibrate improves lipid abnormalities partly via inhibition of TG production and partly via clearance of TG-rich apoB particles by elevating LPL and reduced apoCIII. The prevention of obesity development occurred via energy expenditure. Fenofibrate-mediated hypolipidemic effects together with improved insulin sensitivity and loss of adiposity led to the reductions in the aortic lipid deposition by inhibiting early stages of atherosclerosis possibly via vascular cell adhesion molecule-1 (VCAM-1) modulation. These results suggest that potent PPAR-alpha activators may be useful in the treatment of syndrome X.

Adiposity↗
PubMed2004

Biochemical examination of fibroblasts in the diagnosis and research of oxidative phosphorylation (OXPHOS) defects.

The oxidative phosphorylation system (OXPHOS) is organized in five multi-protein complexes, comprising four complexes (I-IV) of the respiratory chain and ATP synthase (complex V). OXPHOS has a vital role in cellular energy metabolism and ATP production. Enzyme analysis of individual OXPHOS complexes in a skeletal muscle biopsy remains the mainstay of the diagnostic process for patients suspected of mitochondrial cytopathy. A fresh muscle biopsy is preferable to a frozen muscle biopsy because of the possibility to measure the overall capacity of the OXPHOS system. In about 25% of patients referred to our center for muscle biopsy, reduced substrate oxidation rates and ATP + creatine phosphate production rates were found without any defect in complex I-V and the pyruvate dehydrogenase complex. In a subset of patients it is necessary to investigate fibroblasts for diagnostic purposes. The indications for biochemical investigations in fibroblasts are: (a) If no muscle sample is available; (b) If prenatal diagnosis is required; (c) To clarify the results obtained in muscle tissue if no clear-cut diagnosis can be made; (d) If molecular-genetic investigations are required; (e) For research purposes. Fibroblasts are less suitable than fresh muscle for investigating respiratory chain disorders, for the following reasons: (i) A defect that is present in a muscle is not always expressed in fibroblasts. (ii) Exclusion of a defect in fibroblasts does not exclude the diagnosis with regard to muscle. (iii) A specific pattern of abnormalities demonstrated in fibroblasts may not be reflected in muscle tissue. (iv) Enzyme deficiencies found in muscle are generally more pronounced than in fibroblasts. An exact diagnosis of respiratory chain defects is a prerequisite for rational therapy and genetic counseling. Provided guidelines for specimen collection are followed, there are now reliable methods for identifying respiratory chain defects.

Journal Article↗
PubMed2006

Altered cardiac metabolic phenotype after prolonged inhibition of NO synthesis in chronically instrumented dogs.

Acute inhibition of nitric oxide (NO) synthase causes a reversible alteration in myocardial substrate metabolism. We tested the hypothesis that prolonged NO synthase inhibition alters cardiac metabolic phenotype. Seven chronically instrumented dogs were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1) po) for 10 days to inhibit NO synthesis, and seven were used as controls. Cardiac free fatty acid, glucose, and lactate oxidation were measured by infusion of [(3)H]oleate, [(14)C]glucose, and [(13)C]lactate, respectively. After 10 days of L-NAME administration, despite no differences in left ventricular afterload, cardiac O(2) consumption was significantly increased by 30%, consistent with a marked enhancement in baseline oxidation of glucose (6.9 +/- 2.0 vs. 1.7 +/- 0.5 micromol.min(-1).100 g(-1), P < 0.05 vs. control) and lactate (21.6 +/- 5.6 vs. 11.8 +/- 2.6 micromol.min(-1).100 g(-1), P < 0.05 vs. control). When left ventricular afterload was increased by ANG II infusion to stimulate myocardial metabolism, glucose oxidation was augmented further in the L-NAME than in the control group, whereas free fatty acid oxidation decreased. Exogenous NO (diethylamine nonoate, 0.01 micromol.kg(-1).min(-1) iv) could not reverse this metabolic alteration. Consistent with the accelerated rate of carbohydrate oxidation, total myocardial pyruvate dehydrogenase activity and protein expression were higher (38 and 34%, respectively) in the L-NAME than in the control group. Also, protein expression of the constitutively active glucose transporter GLUT-1 was significantly elevated (46%) vs. control. We conclude that prolonged NO deficiency causes a profound alteration in cardiac metabolic phenotype, characterized by selective potentiation of carbohydrate oxidation, that cannot be reversed by a short-term infusion of exogenous NO. This phenomenon may constitute an adaptive mechanism to counterbalance cardiac mechanical inefficiency.

Adaptation, Physiological↗
PubMed1981

Induction of urea cycle enzymes of rat liver by amino acids.

To determine which amino acids in a high casein diet are responsible for induction of the five urea cycle enzyme activities in rat liver, we tube-fed 21 L-amino acids singly to rats over 2 days at maximum doses which did not cause toxicity. The results were compared with the 1.3- to 1.9-fold increases (units/100 g rat) obtained by tube-feeding 2 g N/kg for 2 days as casein hydrolysate. Ala (2 g N/kg), Gly /2 g N/kg), Met (0.2--0.4 g N/kg) and Cys (0.4 g N/kg) were the only amino acids which increased all five activities. Moreover, Met. Ala, Gly and casein hydrolysate in these doses increased immuno-precipitable arginase as much as they increased its activity. A combination of Met, Ala and Gly (2 g N/kg) increased all five activities more than 2 g N/kg of casein hydrolysate. Met (0.05 g N/kg) + Ala (0.08 g N/kg) + Gly (0.1 g N/kg), the amounts of these contained in 2 g N/kg of casein, increased all five enzymes in 2 days as much as this dose of casein hydrolysate. Met (0.06 g N/kg) alone increased all five activities (units/100 g rat) 1.2 to 1.4-fold over controls by increasing g liver/100 g rat. Ammonium citrate or acetate tube-feedings over 8 days at 2 g N/kg increased only AS. The keto-acid of alanine, pyruvate, or the alpha-hydroxy acid of methionine did not increase any enzyme whereas the same molar dose of their amino acids increased all five activities. Thus three amino acids of casein, Ala, Gly and especially Met, account for the enzyme adaptation of the urea cycle on a high casein diet.

Alanine↗
PubMed2003

Apicomplexan parasites contain a single lipoic acid synthase located in the plastid.

Apicomplexan parasites contain a vestigial plastid called apicoplast which has been suggested to be a site of [Fe-S] cluster biogenesis. Here we report the cloning of lipoic acid synthase (LipA) from Toxoplasma gondii, a well known [Fe-S] protein. It is able to complement a LipA-deficient Escherichia coli strain, clearly demonstrating that the parasite protein is a functional LipA. The N-terminus of T. gondii LipA is unusual with respect to an internal signal peptide preceding an apicoplast targeting domain. Nevertheless, it efficiently targets a reporter protein to the apicoplast of T. gondii whereas co-localization with the fluorescently labeled mitochondrion was not detected. In silico analysis of several apicomplexan genomes indicates that the parasites, in addition to the presumably apicoplast-resident pyruvate dehydrogenase complex, contain three other mitochondrion-localized target proteins for lipoic acid attachment. We also identified single genes for lipoyl (octanoyl)-acyl carrier protein:protein transferase (LipB) and lipoate protein ligase (LplA) in these genomes. It thus appears that unlike plants, which have only two LipA and LipB isoenzymes in both the chloroplasts and the mitochondria, Apicomplexa seem to use the second known lipoylating activity, LplA, for lipoylation in their mitochondrion.

Amino Acid Sequence↗
PubMed1987

Fatty acid synthesis and triacylglycerol accumulation in rat liver after chronic ethanol consumption.

1. Liver slices from chronically alcohol-fed rats incubated with 3H2O showed less than half the fatty acid synthesis rates of pair-fed controls. Addition of 50 mmol/l ethanol or of 10 mmol/l lactate and 1 mmol/l pyruvate to the incubation medium did not alter the fatty acid synthesis rates in either groups. Hepatic fatty acid synthesis rates measured in vivo with 3H2O were also significantly reduced in alcohol-fed rats. 2. Time-course experiments showed that after 1 week on the ethanol diet hepatic fatty acid synthesis rates in vitro were similar to control rats, although the liver triacylglycerol content was significantly increased. From the second week of feeding, fatty acid synthesis rates were significantly lower in alcohol-fed rats and the liver triacylglycerol content progressively increased compared with controls. 3. Fatty acid synthase activity in liver cytosolic fractions were similar to controls in the alcohol-fed group after 1 week of feeding but were significantly lower in alcohol-fed rats from the second week onwards. 4. These results indicate that hepatic triacylglycerol accumulation after alcohol feeding is not due to increased fatty acid synthesis. The reduced fatty acid synthesis observed is a consequence of triacylglycerol accumulation.

Animals↗
PubMed1990

Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia.

Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I(50) to susceptible I(50)) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [(14)C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K(m) values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.

Journal Article↗
PubMed1982

Activities of muscle energy supplying enzymes after 5 days complete fasting in young men.

The effect of 5 days' complete fasting on the activity of 7 enzymes of energy supplying metabolism was studied in the vastus lateralis muscles of 9 healthy male volunteers. There was a significant decrease of lactate dehydrogenase (by 66%), triosephosphate dehydrogenase (by 61%), malate:NAD dehydrogenase (by 48%), hexokinase (by 40%), 3-hydroxyacyl-CoA-dehydrogenase (by 40%), triosephosphate dehydrogenase/3-hydroxyacyl-CoA-dehydrogenase (by 35%), citrate synthase (by 33%). Glycerolphosphate:NAD dehydrogenase activity did not decrease significantly. These findings suggest a) that utilization of the major energy substrates decreases; b) that the decrease mainly concerns the catabolism of carbohydrates, that of fatty acids to a lesser extent; c) that the decreased capacity of carbohydrate catabolism is partly compensated for by the predominance of the more economic aerobic-oxidative pathway; d) that the reducing equivalents formed in the cytosol may be transported via the glycerolphosphate shuttle into the respiratory chain to a greater extent, so that a greater portion is not lost by the reduction of pyruvate to lactate.

Adult↗
PubMed1989

Study on ATP-generating system and related hexokinase activity in mitochondria isolated from undifferentiated or differentiated HT29 adenocarcinoma cells.

The functional properties of mitochondria bound hexokinase are compared in two subpopulations of the HT29 human colon cancer cell-line: (1) the HT29 Glc+ cells, cultured in the presence of glucose, which are poorly differentiated and highly glycolytic and (2) the HT29 Glc- cells, adapted to grow in a glucose-free medium, which are 'enterocyte-like' differentiated and less glycolytic when given glucose (Zweibaum et al. (1985) J. Cell Physiol. 122, 21-28). The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase are found to be twice as high in Glc+ cells when compared to Glc- cells. Besides, the respiration rate is decreased in Glc+ cells compared to Glc- cells. These results correlate with the higher glycolytic rate in Glc+ cells. In many tissues, it has been shown that the binding of hexokinase to the mitochondrial outer membrane allows a preferential utilization of the ATP generated by oxidative phosphorylation which, in turn, is activated by immediate restitution of ADP. In highly glycolytic cancer cells, although a large fraction of hexokinase is bound to the mitochondria, the existence of such a channeling of nucleotides is still poorly documented. The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from both Glc+ and Glc- cells either with exogenous ATP or with ATP generated by mitochondria supplied with ADP and succinate (endogenous ATP). Diadenosine pentaphosphate (Ado2P5), oligomycin and carboxyatractyloside (CAT) were used in combination or separately as metabolic inhibitors of adenylate kinase, ATP synthase and ATP/ADP translocator, respectively. Exogenous ATP appears to be 6.5-times more efficient than endogenous ATP in supporting hexokinase activity in the mitochondria from Glc+ cells and only 1.8-times cells. The rate of oxidative phosphorylation being higher in mitochondria from Glc- cells, hexokinase activity is higher in this model when ATP is generated by respiration. Furthermore, in Glc+ mitochondria, the adenylate kinase reaction appears to be an important source of endogenous ATP for bound hexokinase, while, in Glc- mitochondria, hexokinase activity is almost totally dependent on the ATP generated by oxidative phosphorylation. This result might be explained by our previous finding that mitochondria from Glc+ cells lack contact sites between outer and inner membrane, whereas numerous contacts were observed in mitochondria from Glc- cells (Denis-Pouxviel et al. (1987) Biochim. Biophys. Acta 902, 335-348).

Adenocarcinoma↗
PubMed1974

Subcellular localization of isoleucine-valine biosynthetic enzymes in yeast.

By using the method of stepwise homogenization of yeast spheroplast lysates employed previously with the leucine biosynthetic enzymes, it is shown that threonine deaminase, acetohydroxy acid synthase, Mg(2+)-dependent isomero-reductase, and dihydroxy acid dehydratase are particulate. Density gradient centrifugation and the behavior of marker enzymes suggest that all of the above enzymes of the isoleucine-valine biosynthetic pathway are associated with the mitochondria.

Cell-Free System↗
PubMed1985

Pathway of ammonium assimilation in Streptomyces venezuelae examined by amino acid analyses and 15N nuclear magnetic resonance spectroscopy.

To obtain information on the route(s) of ammonium assimilation in Streptomyces venezuelae, cell suspensions transferred to fresh medium lacking nitrogen were pulsed with [15N2]ammonium sulphate. Cells and extracellular fluids were examined by nuclear magnetic resonance and amino acid analysis to assess changes in amino acid pools and the disposition of [15N]ammonium. Following addition of [15N]ammonium, glutamate--glutamine pools of low cell density replacement cultures expanded rapidly and became progressively labelled with 15N, whereas the alanine pool size increased much more slowly and became labelled with 15N to a much lesser extent. These results are consistent with the assimilation of ammonium via glutamate dehydrogenase or glutamine synthetase--glutamate synthase rather than alanine dehydrogenase. Under anaerobic conditions, S. venezuelae assimilates ammonium into alanine rather than glutamate--glutamine. Alanine dehydrogenase may thus function as a vehicle to regenerate NAD+ to maintain substrate-level phosphorylation during periods of anaerobiosis.

Alanine↗
PubMed1998

Cardiodepression by tumor necrosis factor-alpha.

Cardiodepressant effects of tumor necrosis factor-alpha (TNF-alpha have been documented in numerous experimental settings in vivo and in vitro. In vivo administration of TNF-alpha mimicks the cardiovascular pattern of sepsis including septic cardiomyopathy. Serum levels of TNF-alpha were found to be elevated both in sepsis and in numerous non-septic heart disorders. Although an involvement of TNF-alpha in the pathogenesis of septic cardiomyopathy seems likely, presently no definite conclusion can be drawn with regard to the role of TNF-alpha in chronic heart failure. The origin and trigger mechanisms for the release of TNF-alpha in heart failure are a matter of debate, endotoxin (LPS) from intestinal translocation in venous congestion being one possible player. The negative inotropic impact of TNF-alpha is frequently ascribed to the induction of inducible nitric oxide (NO) synthase (iNOS). Results from in vitro studies rather suggest a complex interaction of TNF-alpha with the heart, with pleiotropic effects on cardiomyocyte performance, including an induction of iNOS at higher TNF-alpha concentrations, but NO-independent cardiodepression at low, pathophysiologically more relevant concentrations. TNF-alpha effects on the heart also vary with regard to the kinetics of the process: rapidly occuring cardiodepressant effects include a release of sphingosine and a suppression of the calcium transient, while chronic administration of TNF-alpha was shown to depress the synthesis of precursors for the phosphoinositide pathway and inhibit pyruvate dehydrogenase activity and mitochondrial function. Whether secondary cytokines induced by TNF-alpha in cardiomyocytes contribute to cardiodepression or whether apoptotic signals activated by TNF-alpha are involved in the cardiodepressive pathways is presently unknown.

Animals↗
PubMed1992

Glucose 1,6-bisphosphate-overloaded erythrocytes: a strategy to investigate the metabolic role of the bisphosphate in red blood cells.

Human erythrocytes overloaded with glucose 1,6-bisphosphate were prepared in order to establish the metabolic significance of this phosphorylated sugar in the intact red cell. The intracellular glucose 1,6-bisphosphate concentration was increased six- and twofold over the normal level by encapsulating (i) the commercially available compound and (ii) the glucose 1,6-bisphosphate synthase obtained from rabbit skeletal muscle, respectively. In both experimental conditions, a reduction of glucose utilization by the loaded cells was observed after reequilibration to the steady state. At the steady state, the concentrations of the glycolytic intermediates and of the adenine nucleotides appeared substantially unmodified when compared with those of controls, with the exception of a 50% reduction of glucose and fructose 6-phosphate measured in erythrocytes encapsulated with exogenous glucose 1,6-bisphosphate. Under the considered experimental conditions, the elevated intracellular glucose 1,6-bisphosphate appears to display an inhibitory effect on hexokinase that overcomes the possible activation of phosphofructokinase or pyruvate kinase.

2,3-Diphosphoglycerate↗
PubMed2004

Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli.

The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (aspartate ammonia-lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of aspartate to fumarate and ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine beta-lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.

Aspartic Acid↗
PubMed2006

Phosphate analogues as probes of the catalytic mechanisms of MurA and AroA, two carboxyvinyl transferases.

The role in catalysis of phosphate with AroA (enolpyruvyl shikimate 3-phosphate synthase) and MurA (enolpyruvyl UDP-GlcNAc synthase) was probed using phosphate analogues and an AroA mutant. Phosphate, the second reaction product, increases the reactivity of the enolpyruvyl products (EP-OR's) approximately 10(5)-fold in the reverse reaction, forming phosphoenolpyruvate and R-OH (shikimate 3-phosphate or UDP-GlcNAc). Phosphate is intrinsically unreactive with EP-OR, raising the question of how AroA and MurA promote EP-OR reactivity. Eleven phosphate analogues were examined. All those with tetrahedral geometries bound with AroA, except sulfate, while no nontetrahedral analogues did. Arsenate, vanadate, and fluorophosphate caused reactions of AroA and MurA with EP-OR's, yielding pyruvate and R-OH. Their k(cat)/K(M) values relative to phosphate were similar for both enzymes, ca. 100-fold worse for arsensate, 200-fold worse for vanadate, and 5000-fold worse for fluorophosphate, implying similar interactions with both enzymes. Examination of the arsenate-promoted reactions using [3'-(3)H]EP-OR's, (2)H(2)O, and H(2)(18)O provided evidence of an arseno-tetrahedral intermediate, analogous to the natural tetrahedral intermediate, proceeding to arsenoenolpyruvate, which spontaneously broke down to pyruvate and arsenate. The only physicochemical property that appeared to be essential for reactivity of the analogues was the presence of a proton. Titration of the intrinsic tryptophan fluorescence of the weakly active AroA mutant, Asp313Ala (D313A), demonstrated a fluorescence decrease upon enolpyruvyl shikimate 3-phosphate (EPSP) binding, and a further decrease upon binding of phosphate or arsenate to AroA_D313A.EPSP, suggesting a further conformational change. We are hopeful that understanding enzyme-phosphate interactions will make it possible to design inhibitors that can use the high endogenous phosphate concentration in bacteria to enhance inhibitor binding.

Binding Sites↗
PubMed2002

Ethyl pyruvate modulates inflammatory gene expression in mice subjected to hemorrhagic shock.

Administration of pyruvate, an effective scavenger of reactive oxygen species, has been shown to be salutary in numerous models of redox-mediated tissue or organ injury. Pyruvate, however, is unstable in solution and, hence, is not attractive for development as a therapeutic agent. Herein, ethyl pyruvate, which is thought to be more stable than the parent compound, was formulated in a calcium-containing balanced salt solution [Ringer ethyl pyruvate solution (REPS)] and evaluated in a murine model of hemorrhagic shock and resuscitation (HS/R). Resuscitation with REPS instead of Ringer lactate solution (RLS) significantly improved survival at 24 h and abrogated bacterial translocation to mesenteric lymph nodes and the development of increased ileal mucosal permeability to FITC-labeled dextran (4,000 Da) at 4 h. Mice treated with REPS instead of RLS also had lower circulating levels of alanine aminotransferase at 4 h. Treatment with REPS instead of RLS decreased activation of nuclear factor-kappaB in liver and colonic mucosa after HS/R and also decreased the expression of inducible nitric oxide synthase, tumor necrosis factor, cyclooxygenase-2, and interleukin-6 mRNA in liver, ileal mucosa, and/or colonic mucosa. These data support the view that resuscitation with REPS modulates the inflammatory response and decreases hepatocellular and gut mucosal injury in mice subjected to HS/R.

Animals↗
PubMed2005

Role of lipids in the early developmental stages of experimental immune diabetes induced by multiple low-dose streptozotocin.

The present work examines the role of lipids in the development of the Type 1 diabetes induced by the administration of multiple low doses of streptozotocin (STZ) in C57BL/6J mice. The study was performed before and after the onset of clear hyperglycemia, and the results were as follows. First, 6 days after the first dose of STZ, while plasma glucose and insulin levels remained similar to those observed in the control mice, plasma free fatty acid (FFA) levels were significantly increased (P < 0.05). At that time, a marked increase of triglyceride content in gastronemius muscle was accompanied by a diminished activity of pyruvate dehydrogenase complex, suggesting an impaired glucose oxidation. Furthermore, a decrease of both triglyceride content and lipoprotein lipase activity was observed in the epididymal fat tissue. Second, 12 days after the first injection of STZ, hyperglycemia was accompanied by hypertriglyceridemia, a more pronounced increase of plasma FFA, and a significant (P < 0.05) reduction of insulinemia. At this time, both the adipose tissue and the gastrocnemius muscle showed a further deterioration of all parameters mentioned after 6 days. Moreover, in the gastrocnemius muscle, an impaired nonoxidative pathway of glucose metabolism was observed [significant reduction (P < 0.05) of glycogen mass, glucose-6-phosphate content, and glycogen synthase activities] at this time point. Finally, the data suggest for the first time that, in mice, Type 1 diabetes induced by multiple low doses of STZ and enhanced lipolysis of fat pads leads to an increase in the availability of plasma FFA, which seems to play a role in the early steps of diabetes evolution.

Animals↗