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At least 883 records · Page 49Linked to original sources
PubMed1994

Muscle mitochondrial ATP production in progressive supranuclear palsy.

Six patients with progressive supranuclear palsy (PSP) and 12 age-matched disease-free subjects participated in this study designed to compare rates of ATP production by intact mitochondria from biopsied skeletal muscle. When pyruvate and malate were used as metabolic substrates, rates of ATP production were 0.184 +/- 0.025 mumol/min/U of citrate synthase (CS) activity (a mitochondrial marker) in control subjects and 0.131 +/- 0.051 mumol/min/U of CS in PSP patients. In the presence of succinate, rates of ATP formation were 0.137 +/- 0.02 mumol/min/U of CS in controls and 0.109 +/- 0.04 mumol/min/U of CS in patients. With N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ascorbate, rates were 0.034 +/- 0.008 mumol/min/U of CS in controls and 0.022 +/- 0.01 mumol/min/U of CS in PSP subjects. Differences between the control and PSP populations reached statistical significance with pyruvate/malate and TMPD/ascorbate. No differences in either muscle histopathology or histochemistry were found between patient and control subjects. Results of this study suggest that oxidative phosphorylation defects occur in muscle mitochondria from patients with PSP.

Adenosine Triphosphate↗
PubMed2006

Proteome changes after metabolic engineering to enhance aerobic mineralization of cis-1,2-dichloroethylene.

Metabolically engineered Escherichia coli has previously been used to degrade cis-1,2-dichloroethylene (cis-DCE). The strains express the six genes of an evolved toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green, which formed a reactive epoxide) with either (1) gamma-glutamylcysteine synthetase (GSHI, which forms glutathione) and the glutathione S-transferase IsoILR1 from Rhodococcus AD45 (which adds glutathione to the reactive cis-DCE epoxide) or (2) with an evolved epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA F108L/I219L/C248I which converts the reactive cis-DCE epoxide to a diol). Here, the impact of this metabolic engineering for bioremediation was assessed by investigating the changes in the proteome through a quantitative shotgun proteomics technique (iTRAQ) by tracking the changes due to the sequential addition of TOM-Green, IsoILR1, and GSHI and due to adding the evolved EchA versus the wild-type enzyme to TOM-Green. For the TOM-Green/EchA system, 8 proteins out of 268 identified proteins were differentially expressed in the strain expressing EchA F108L/I219L/C248I relative to wild-type EchA (e.g., EchA, protein chain elongation factor EF-Ts, 50S ribosomal subunits L7/L12/L32/L29, cysteine synthase A, glycerophosphodiester phosphodiesterase, iron superoxide dismutase). For the TOM-Green/IsoILR1/GSHI system, the expression level of 49 proteins was changed out of 364 identified proteins. The induced proteins due to the addition of TOM-Green, IsoILR1, and GSHI were involved in the oxidative defense mechanism, pyruvate metabolism, and glutathione synthesis (e.g., 30S ribosomal subunit proteins S3 and S16, 50S ribosomal subunit protein L20, alkyl hydroperoxide reductase, lactate dehydrogenase, acetate kinase, cysteine synthase A). Enzymes involved in indole synthesis, fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle were repressed (e.g., tryptophanase, acetyl-CoA carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase). Hence, the metabolic engineering that leads to enhanced aerobic degradation of 1 mM cis-DCE (2.4-4-fold more chloride ions released) and reduced toxicity from cis-DCE epoxide results in enhanced synthesis of glutathione coupled with an induced stress response as well as repression of fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle.

Aerobiosis↗
PubMed1995

Lipid metabolism and membrane composition are altered in the brains of type II diabetic mice.

CBL/57 strain db/db mice exhibit type II (noninsulin-dependent) diabetes. The affected mice are markedly hyperinsulinemic, hyperglycemic, and hypercholesterolemic, and their serum K+ levels are decreased. The brains of the diabetic mice are significantly smaller than those of their lean, control littermates, but the protein concentration is normal. The low brain weight is accompanied by a loss of major fatty acid components within the whole brain, nerve endings, and mitochondrial membranes. Cholesterol levels are low in whole brain but are not significantly different from normal in the synaptosomal membranes. The phospholipid concentration is significantly decreased in whole brain homogenates, crude synaptosomal membranes, and crude mitochondrial membranes of the diabetic mice. In addition, the specific activities of membrane-bound synaptosomal acetylcholinesterase, Na+,K(+)-ATPase, and Mg(2+)-ATPase are decreased in crude synaptosomal membranes of the diabetic mice. The specific activities of carnitine palmitoyltransferase I and carnitine acetyltransferase are significantly increased in the crude mitochondrial fraction isolated from the brains of the type II diabetic mice, whereas the specific activity of pyruvate dehydrogenase complex is decreased. The specific activities of two other mitochondrial enzymes--monoamine oxidase B and citrate synthase--and a cytosolic enzyme--lactate dehydrogenase--are unaltered. The ability to synthesize cyclic AMP is markedly decreased in the brains of the diabetic mice. The concentrations of carnitine and of the amino acids, glutamate, aspartate, glutamine, and serine are unaltered, whereas glycine levels are significantly elevated in the brains of the db/db mice. The data suggest that in vivo the brains of the diabetic mice exhibit a decreased capacity for glucose oxidation and increased capacity for fatty acid oxidation. This hypothesis is supported by the finding that cerebral mitochondria isolated from the db/db mice oxidize [1-14C]palmitate to 14CO2 at a rate almost twice that of control mitochondria. The present findings emphasize the potentially serious alteration of brain metabolism in uncontrolled type II diabetes.

Acetylcholinesterase↗
PubMed1994

Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield.

Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). It has been shown that overexpression of transketolase increases the production of DAHP in an aroB mutant strain (unable to further metabolize DAHP) with elevated DAHP synthase. However, the yield (percent conversion) of DAHP from glucose is still low. Stoichiometric analysis shows that many enzymes compete for intracellular PEP. In particular, the phosphotransferase system, responsible for glucose transport in E. coli, uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. This stoichiometric limitation greatly reduces the yield of aromatic metabolites. To relieve this limitation, we overexpressed PEP synthase in the presence of glucose and showed that it increased the final concentration and the yield of DAHP by almost twofold, to a near theoretical maximum. The PEP synthase effect is not observed without overproduced transketolase, suggesting that erythrose 4-phosphate is the first limiting metabolite. This result demonstrates the utility of pathway analysis and the limitation of central metabolites in the high-level overproduction of desired metabolites.

Culture Media↗
PubMed2002

Lipid oxidation fuels recovery from exhaustive exercise in white muscle of rainbow trout.

The oxidative utilization of lipid and carbohydrate was examined in white muscle of rainbow trout (Oncorhynchus mykiss) at rest, immediately after exhaustive exercise, and for 32-h recovery. In addition to creatine phosphate and glycolysis fueling exhaustive exercise, near maximal activation of pyruvate dehydrogenase (PDH) at the end of exercise points to oxidative phosphorylation of carbohydrate as an additional source of ATP during exercise. Within 15 min postexercise, PDH activation returned to resting values, thus sparing accumulated lactate from oxidation. Glycogen synthase activity matched the rate of glycogen resynthesis and represented near maximal activation. Decreases in white muscle free carnitine, increases in long-chain fatty acyl carnitine, and sustained elevations of acetyl-CoA and acetyl carnitine indicate a rapid utilization of lipid to supply ATP for recovery. Increases in malonyl-CoA during recovery suggest that malonyl-CoA may not regulate carnitine palmitoyltransferase-1 in trout muscle during recovery, but instead it may act to elongate short-chain fatty acids for mitochondrial oxidation. In addition, decreases in intramuscular triacylglycerol and in plasma nonesterified fatty acids indicate that both endogenous and exogenous lipid fuels may be oxidized during recovery.

Adenosine Diphosphate↗
PubMed1990

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts.

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.

Cell Differentiation↗
PubMed1989

Age-related modification of enzyme activities in synaptosomes isolated from rat cerebral cortex.

In synaptosomes from rat cerebral cortex, the potential catalytic activity of some enzymes related to energy metabolism--namely, phosphofructokinase and citrate synthase--is not affected by aging. In contrast, the maximum velocity (Vmax) of cytochrome oxidase and of pyruvate dehydrogenase decreases in aged rats. A marked increase is found in the Vmax of glucose 6-phosphate dehydrogenase in aged rats and could be related to the availability of NADPH for antiperoxidative processes. Pretreatments of experimental animals with certain drugs were done to investigate the plasticity of enzyme proteins during aging. Papaverine, which acts on macrocirculation, is ineffective, but delta-yohimbine acting on microcirculation and metabolism and almitrine acting on oxygen availability both could interfere with the potential activity of some enzymes. However, their influence differs with the age of the rats.

Aging↗
PubMed1982

Inhibition of medium and short-chain fatty acid oxidation in rat heart mitochondria by dichloroacetate.

The effect of dichloroacetate (DCA) on the oxidation of medium-chain and short-chain fatty acids was studied in rat heart mitochondria. Dichloroacetate (1-10 mM) markedly decreased medium-chain and the short-chain fatty acid oxidation (oxygen uptake and release of 14CO2 from labelled fatty acids) by heart mitochondria; the oxidation of long-chain fatty acid, however, was unaffected by the compound. THe oxidation of acylcarnitines was not influenced by DCA. Dichloroacetate inhibited medium-chain and short-chain fatty acid oxidation by inhibiting the activity of mitochondrial acyl-CoA synthases. Inhibition of the enzyme activity by DCA was rapid and not due to its effect on pyruvate dehydrogenase.

Acetates↗
PubMed2005

Structural characterization of sialic acid synthase by electrospray mass spectrometry--a tetrameric enzyme composed of dimeric dimers.

Sialic acid synthase (NeuB) encoded by the neuB gene catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form N-acetylneuraminic acid. The enzyme is essential for the biosynthesis of polysialic acid, a capsular sugar polymer functioning as a virulent factor and antiphagocytic barrier. This report demonstrates the first characterization on the quaternary structure of NeuB from Escherichia coli (EcNeuB) and Streptococcus agalactiae (SaNeuB) by nanoflow electrospray ionization mass spectrometry (ESI-MS). Under non-denaturing conditions, Tris buffer was observed to induce a higher ratio of tetramer/dimer of NeuB in the ESI mass spectra, providing supportive evidence for the existence of a "structurally-specific" tetramer. The instrument parameters were found to significantly affect the ratio of detected tetramer/dimer in ESI mass spectra. The harshest conditions, including high desolvation voltages and pressure in the collision cell, led to enhanced detection of the 160 kDa tetramer. The prevalence of dimeric form is likely the cause in loss of tetramer stability in gas-phase arising from insufficient collisional cooling, which implies an asymmetric assembly, possibly composed of dimeric dimers. Most interestingly, the hypothesis was further supported by chemical cross-linking of SaNeuB, in which the reaction of shorter linker yielded mainly the dimer whereas that of longer linker produced both dimer and tetramer. Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis.

Dimerization↗
PubMed2005

Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation.

The development of age-related proliferative disorders of the prostate gland is supported by transdifferentiation and cellular senescence processes in the stroma. Both processes are involved in remodeling of stromal tissue, as observed in benign prostatic hyperplasia (BPH), and in "reactive stroma" adjacent to prostate cancer (PCa). It has been assumed that TGF-beta1 plays a key role in the aging prostate by inducing premature senescence and favoring myofibroblast differentiation. Therefore, we evaluated the stromal cell phenotypes of human primary adult prostatic fibroblasts (n=3) and the molecular and cellular mechanisms of growth arrest after treatment with TGF-beta1 and of in vitro cellular senescence. Microarray analysis, quantitative PCR, immunofluorescence and western blot revealed that cellular senescence and transdifferentiation of fibroblasts have distinct underlying mechanisms, pathways and gene and protein expression profiles in human PrSCs. In clear contrast to senescent cells, TGF-beta1-treated cells morphologically transdifferentiated into myofibroblasts with dense cytoskeletal fibers and increased expression of smooth muscle cell alpha-actin, calponin and tenascin. TGF-beta1 induced neither expression of senescence-associated markers nor genes involved in terminal growth arrest, such as senescence-associated beta-galactosidase and cyclin-dependent kinase (cdk) inhibitors p16(Ink4A) and p21(Cip1) but increased p15(Ink4B) protein expression. Differentiation inhibitor (Id-1) protein level down-regulation was observed under both conditions. Genes specifically up-regulated by transdifferentiation but not by cellular senescence of PrSCs were metalloproteinase 1 tissue inhibitor (Timp1), transgelin (Tagln), gamma 2 actin (Actg2), plasminogen activator inhibitor 1 (Serpinel), insulin-like growth factor binding protein 3 (Igfbp3), parathyroid hormone-like hormone (Pthlp), Tgfb-1, four and a half LIM domains 2 (Fhl-2), hydrogen peroxide-inducible clone 5 (Hic5) and cartilage oligomeric matrix protein (Comp). Other genes, such as Cdc28 protein kinase 1 (Cks1b), v-myb myeloblastosis viral oncogene homolog (MybL2), pyruvate kinase, muscle 2 (Pkm2) and Forkhead box M1 (FoxM1), were down-regulated only upon TGF-beta1 treatment but not by cellular senescence. Pyruvate dehydrogenase kinase 3 (Pdk3) and connective tissue growth factor (Ctgf) were up-regulated and hyaluronan synthase 3 (Has3) down-regulated under both conditions. Moreover, GageC1, a prostate/testis-specific protein overexpressed in symptomatic BPH and PCa was induced in transdifferentiated stromal cells. Genes such as GageC1 could be promising targets for therapeutic inhibitors of stromal tissue remodeling and progression of BPH and PCa.

Cell Differentiation↗
PubMed1984

Glycolate metabolism is under nitrogen control in chlorella.

The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR).During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate.

Journal Article↗
PubMed2003

The effect of crude and purified Cerastes vipera venom protein fractions on respiratory chain function in cultured human fibroblasts.

This study was conducted to investigate the effect of crude and venom protein fractions of Cerastes vipera on the major enzymatic components of the respiratory chain in cultured human fibroblasts. Cerastes vipera crude venom was fractionated into seven protein fractions by using 8% preparative native polyacrylamide gel electro-phoresis. The effect of crude venom and purified venom fractions (F1-F7) on respiratory chain function in cultured human fibroblasts was investigated. Confluent fibroblast cultures were incubated with crude venom and venom protein fractions for a fixed period of three hours at 37 degrees C. Crude venom and venom protein fractions 2, 4, 5 and 6 significantly increased the production of lactate as compared to control fibroblasts. The production of pyruvate was markedly decreased in all these cell lines. The ratio of lactate/pyruvate was increased in the cells and in the culture medium by crude venom and venom protein fractions 2, 4, 5 and 6. On the other hand fractions 1,3 and 7 exhibited no effect in relation to lactate and pyruvate production. Similarly the crude venom protein and venom protein fractions 2, 4, 5 and 6 significantly lowered the activity of the major enzymatic component of the respiratory chain in fibroblast mitochondria. In conclusion it is apparent that the crude and venom protein fractions 2, 4, 5 and 6 resulted in a significant lowering of the mitochondrial respiratory chain activity in cultured human fibroblasts.

Animals↗
PubMed1990

Modified branched-chain amino acid pathways give rise to acyl acids of sucrose esters exuded from tobacco leaf trichomes.

A major diversion of carbon from branched-chain amino acid biosynthesis/catabolism to form acyl moieties of sucrose esters (6-O-acetyl-2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-beta-D- fructofuranosides) was observed to be associated with specialized trichome head cells which secrete large amounts of sucrose esters. Surface chemistry and acetyl and acyl substituent groups of tobacco (T.I. 1068) sucrose esters were identified and quantified by gas chromatography/mass spectrometry. Sucrose esters were prominent surface constituents and 3-methylvaleric acid, 2- and 3-methylbutyric acid, and methylpropionic acid accounted for 60%, 25% and 9%, respectively, of total C3--C7 acyl substituents. Radiolabeled Thr, Ile, Val, Leu, pyruvate and Asp, metabolites of branched-chain amino acid pathways, were compared with radioactively labeled acetate and sucrose as donors of carbon to sucrose, acetyl and acyl components of sucrose esters using epidermal peels with undisturbed trichomes. Preparations of biosynthetically competent trichome heads (site of sucrose ester formation) were also examined. Results indicate that 3-methylvaleryl and 2-methylbutyryl groups are derived from the Thr pathway of branched-chain amino acid metabolism, 3-methylbutyryl and methylpropionyl groups are formed via the pyruvate pathway, and that acetyl groups are principally formed directly via acetyl-CoA. Arguments are presented which rule out participation of fatty acid synthase in the formation of prominent acyl acids. Results suggest that the shunting of carbon away from the biosynthesis of Val, Leu and Ile may be due to a low level of amino acid utilization in protein synthesis in specialized glandular head cells of trichomes. This would result in the availability of corresponding oxo acids for CoA activation and esterification to form sucrose esters. Preliminary evidence was found for the involvement of cycling reactions in oxo-acid-chain lengthening and for utilization of pyruvate-derived 2-oxobutyrate to form straight-chain acyl substituents.

Acyl Coenzyme A↗
PubMed1997

Mechanisms by which carbohydrates regulate expression of genes for glycolytic and lipogenic enzymes.

Regulation of gene expression by nutrients is an important mechanism in the adaptation of mammals to their nutritional environment. This is especially true for enzymes involved in the storage of energy, such as the lipogenic and glycolytic enzymes in liver and adipose tissue. Transcription of the genes for lipogenic and glycolytic enzymes is stimulated by glucose in adipose tissue, liver, and pancreatic beta-cells. Several lines of evidence suggest that glucose must be metabolized to glucose-6-phosphate to stimulate gene transcription. In adipose tissue, insulin increases the expression of lipogenic enzymes indirectly by stimulating glucose uptake. In the liver, insulin also acts indirectly by stimulating the expression of glucokinase and, hence, by increasing glucose metabolism. Glucose response elements have been characterized for the L-pyruvate kinase and S14 genes. They have in common the presence of a sequence 5'-CACGTG-3', which binds a transcription factor called USF (upstream stimulatory factor). Another glucose response element, which uses a transcription factor named Sp1, has been characterized in the gene for the acetyl-coenzyme A carboxylase. The mechanisms linking glucose-6-phosphate to the glucose-responsive transcription complex are largely unknown.

Acetyl-CoA Carboxylase↗
PubMed1982

Biosynthesis of aromatic compounds: 13C NMR spectroscopy of whole Escherichia coli cells.

13C and 31P NMR spectra of wild-type Escherichia coli showed resonances from metabolic intermediates of glycolysis and ATP formation but no detectable signals from aromatic amino acids. However, tyrosine biosynthesis from D-[l-13C]glucose was observed in cells harboring a feedback-resistant allele of aroF, the gene encoding tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC4.1.2.15], one of the isoenzymes that control carbon flow through the common aromatic biosynthetic pathway. A similar accumulation of tyrosine and phenylalanine is seen in cells carrying a multiple-copy plasmid that carries a wild-type aroF allele in addition to pheA and tyrA, the structural genes for controlling enzymes of the terminal pathways to phenylalanine and tyrosine biosynthesis. These in vivo measurements by a noninvasive probe suggest feedback inhibition as the quantitatively major mechanism controlling carbon flow in the common aromatic compound biosynthetic pathway. In strains accumulating aromatic amino acids, a transient accumulation of trehalose was detected, indicating that previously unknown changes in Escherichia coli metabolism accompany overproduction of aromatic compounds.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1994

Changes in glycogen metabolism in liver of gold thioglucose injected mice during the development of obesity.

A study of glycogen metabolism in the liver has been carried out in gold thioglucose (GTG) injected mice during the development of obesity. In GTG obese mice, overt obesity, hyperglycaemia and hyperinsulinaemia had developed by 6 weeks after the injection of GTG. Beyond 6 weeks after GTG injection, the gain of body weight and increment in serum glucose and insulin levels with age in obese mice were not obvious when compared with those of age-matched control animals. The glycogen concentration, total glycogen storage, activity of glycogen synthase R and activity of phosphorylase a in the liver from GTG obese mice were significantly greater than those in lean mice from 2-4 weeks after GTG injection and remained higher thereafter. These results demonstrate that the increased liver glycogen storage and increased activity of glycogen synthase and phosphorylase occur early in the development of obesity and at a similar time to previously reported increases in pyruvate dehydrogenase activity (Caterson et al. (1987) Biochem. J. 243, 549-553) and lipid synthesis in liver (Cooney et al. (1989) Biochem. J. 259, 651-657). The emergence of these abnormalities in glycogen metabolism early in the development of obesity may contribute to the establishment of glucose intolerance and insulin resistance in this model of obesity which became apparent at approximately the same time after GTG injection.

Analysis of Variance↗