Citrate Synthase

Background-Although a number of exercise systems have been developed to mitigate the physiological deconditioning that occurs in microgravity, few have the capacity to positively impact multiple physiological systems and still meet the volume/mass requirements needed for missions beyond low earth orbit. The purpose of this study was to test the gravity-independent Multi-Mode Exercise Device (M-MED) for both resistance (RE) and aerobic (AE) training stimuli. Methods-Eight men and nine women (mean age 22.0±0.4 years) completed five weeks of training on the M-MED: RE 4×7 squats two days a week, and AE 4×4-min rowing bouts at ~90% VO 2 max three days a week. Pre-and post-training data collection included an aerobic capacity test, MR imaging, strength testing, and vastus lateralis muscle biopsy. Results-VO 2 max increased 8%, 3RM strength 18%, and quadriceps femoris cross-sectional area (CSA) 10%. Knee extensor strength increased at all isokinetic speeds tested. Subjects also demonstrated improved resistance to fatigue in knee extension. At the cellular and molecular level, the biopsy revealed increases in mixed myofiber CSA (13%), citrate synthase activity (26%), total RNA concentration (24%), IGF-I mRNA (77%), Type IIa Myosin Heavy Chain (MHC) mRNA (8%), and concomitant decrease in Type IIx MHC mRNA (-23%). None of the changes were gender-specific.

NEURO-ONCOLOGY • NOVEMBER 2019 pools respectively in comparison with cells not treated with OAA. M+3 13 C labeled pyruvate entered TCA cycle via acetyl-CoA, where we also observed reduced levels of M+2 13 C labeled citrate (20.5%) and glutamate (23.9%) isotopomers. Pyruvate can also enter TCA cycle via pyruvate carboxylation pathway and this activity was also found to be slightly decreased in the OAA treated cells. All the differences were statistically significant. These results indicate that OAA can be used to alter bioenergetics of GBM cells, specifically glucose oxidation.

SUMMARYThe evolution of metabolic pathways involved in energy production was studied in the flight muscles of 28 species of orchid bees. Previous work revealed that wingbeat frequencies and mass-specific metabolic rates decline in parallel by threefold as body mass increases interspecifically over a 20-fold range. We investigated the correlated evolution of metabolic rates during hovering flight and the flux capacities, i.e. Vmaxvalues, of flight muscle enzymes involved in substrate catabolism, the Krebs cycle and the electron transport chain. Vmax at the hexokinase (HK) step scales allometrically with an exponent almost identical to those obtained for wingbeat frequency and mass-specific metabolic rate. Analysis of this relationship using phylogenetically independent contrasts supports the hypothesis of correlated evolution between HK activity and mass-specific metabolic rate. Although other enzymes scale allometrically with respect to body mass, e.g. trehalase, glycogen phosphoryl...

Capacities and effects of cold or warm acclimation were investigated in two zoarcid species from the North Sea (Zoarces viviparus) and the Antarctic (Pachycara brachycephalum) by investigating temperature dependent mitochondrial respiration and activities of citrate synthase (CS) and NADP + -dependent isocitrate dehydrogenase (IDH) in the liver. Antarctic eelpout were acclimated to 5°C and 0°C (controls) for at least 10 months, whereas boreal eelpout, Z. viviparus (North Sea) were acclimated to 5°C and to 10°C (controls). Liver sizes were found to be increased in both species in the cold, with a concomitant rise in liver mitochondrial protein content. As a result, total liver state III rates were elevated in both cold-versus and warm-exposed P. brachycephalum and Z. viviparus, with the highest rates in boreal eelpout acclimated to 5°C. CS and IDH activities in the total liver were similar in Z. viviparus acclimated to 5°C and 10°C, but decreased in those warm acclimated versus control P. brachycephalum. Enzyme capacities in the total liver were higher in eelpout from Antarctica than those from the North Sea. In conclusion, cold compensation of aerobic capacities in the liver seems to be linked to an increase in organ size with unchanged specific mitochondrial protein content. Despite its life in permanently cold climate, P. brachycephalum was able to reduce liver aerobic capacities in warm climate and thus, displayed a capacity for temperature acclimation.

Citrate synthase (CS) is involved in citric acid biosynthesis which is a well-established metabolic pathway. The condensation of acetyl-CoA with oxaloacetate is catalyzed by CS. Citric acid (CA) has a number of applications in pharmaceutical industry. CA in combination with bicarbonates is used as an effervescent in the preparations of tablets and powders. It has also been used as an anticoagulant and acidulant to form mild astringent. In current study, detailed structural and functional analyses of CS protein were carried out using various bioinformatics tools. Structural modeling was also done by building 3D model of CS from Aspergillus niger ANJ-120 using Modeller 9.16 software. The 3D Model was then evaluated using different online approaches. Furthermore, superimposition of query and template structures, Root Mean Squared Deviation and visualization of generated model were done through UCSF Chimera 1.5.3. Even though various roles of CS protein were already known and verified e...

Partial nucleotide sequences of the citrate synthase (gltA) gene from different rhizobia genera were determined. Tree topologies based on this housekeeping gene were similar to that obtained using 16S rRNA sequences. However gltA appeared to be more reliable at determining phylogenetic relationships of closely related taxa. We propose gltA sequences as an additional tool to be used in molecular phylogenetic studies.

This study focused on strain improvement and fermentation optimization for enhanced citric acid production by Aspergillus niger using waste molasses as a substrate. Fifty strains were isolated from soil samples collected in Lahore, Pakistan, and the most productive strain was identified through cultural characteristics and slide culture confirmation. UV and gamma irradiation were employed for strain improvement, with UV treatment producing the most efficient mutant strain (ISO-18-UV-M-10). A molasses-based fermentation medium was optimized using the following conditions: 30 °C, pH 5.5, 150 g/L initial sugar concentration, and 144 h fermentation time under shake flask conditions. To reduce the inhibitory effects of heavy metals, EDTA and K₄Fe(CN)₆ were tested as chelating agents. The best results were achieved with 150 ppm EDTA added at 72 h and 200 ppm K₄Fe(CN)₆ added at 48 h, respectively. Simultaneous addition of both chelators reduced productivity. The highest citric acid yield reached 70.5 g/L using K₄Fe(CN)₆, representing a 15.67-fold increase over the original wild-type strain (4.5 g/L). Under standard optimized conditions without chelators, the UV mutant produced 53.0 g/L, equivalent to a 10.3-fold increase, establishing the effectiveness of mutagenesis and process optimization in boosting production.

Few cases of infantile liver disease associated with mitochondrial DNA (mtDNA) depletion have been reported. Most of the patients died before 1 year of age of severe liver failure. We describe a new case, a 28-month-old child, presenting with cholestasis at age 2 months, complicated by progressive portal and lobular liver fibrosis. Growth and psychomotor development are undisturbed. There is no clinical evidence of either myopathy or neurological involvement. Metabolic investigation in plasma revealed an abnormal oxido-reduction status after fasting and after carbohydrate-rich meals. Light microscopy performed on liver biopsies revealed steatosis, abnormal hepatocytes with an ''oncocytic'' appearance and extensive fibrosis. Electron microscopic investigation showed an increased number of mitochondria T   of potential causes of cholestasis in early life is long and varied. In addition to the most common causes, such as biliary atresia, metabolic disorders should be investigated, especially when a microvesicular steatosis is associated (1). We know that impairment of mitochondrial functions can induce both necrosis and microvesicular steatosis, leading in some cases to cirrhosis (2,3). Among the possible genetic causes of mitochondrial dysfunction in infants, mitochondrial DNA (mtDNA) depletion has to be considered.

Peripheral arterial disease (PAD) is associated with muscle metabolic changes that may contribute to the disability in these patients. However, the biochemical defects in PAD have not been identified. The present study was undertaken to test the hypothesis that PAD is associated with specific defects in skeletal muscle electron transport chain activity. Seventeen patients with PAD and nine age-matched controls underwent gastrocnemius muscle biopsies. There were no differences in the mitochondrial content per gram of skeletal muscle as assessed by citrate synthase activity between the PAD patients and the control subjects. Skeletal muscle NADH dehydrogenase activity was decreased by 27% compared with controls when expressed per unit of citrate synthase activity. Expression of enzyme activities normalized to cytochrome c-oxygen oxidoreductase activity confirmed a 26% decrease in NADH dehydrogenase activity and also demonstrated a 38% decrease in ubiquinol-cytochrome c oxidoreductase a...

Hepatic mitochondrial and peroxisomal oxidative capacities were studied in young (4-5 weeks old) and adult (6-9 months old) lean and obese ob/ob mice that were fed or starved for 24 or 48 h. The adult obese mice showed elevated capacity for mitochondrial oxidation (ng-atoms of O consumed/min per mg of protein) of lipid and non-lipid substrates, with the exception of pyruvate + malate, and elevated activities of citrate synthase and total carnitine palmitoyltransferase. Oxidative rates and enzyme activities were not affected by starvation of lean or obese mice, and both males and females responded similarly. Peroxisomal palmitoyl-CoA oxidation (nmol/min per mg of peroxisomal protein) was also increased in livers of adult obese mice and did not change with starvation. In young mice, hepatic mitochondrial and peroxisomal oxidative capacities in lean and obese mice were comparable. The increased mitochondrial and peroxisomal oxidative capacities appear to develop with maturation in obes...

Background —Patients with peripheral arterial disease (PAD) have exercise limitation due to claudication-limited pain and metabolic alterations in skeletal muscle. PAD is also associated with oxidative stress, which is a known cause of mitochondrial DNA (mtDNA) injury. The present study was designed to test the hypothesis that PAD is associated with mtDNA injury, as reflected by an increased frequency of a specific 4977–base pair (bp) mtDNA deletion mutation. Methods and Results —The deletion frequency was quantified in gastrocnemius muscle of 8 patients with unilateral PAD and 10 age-matched control subjects with the use of polymerase chain reaction methodologies. Muscle from the hemodynamically unaffected (less affected) PAD limb showed an 8-fold increased deletion frequency and the hemodynamically affected (worse affected) PAD limb had a 17-fold increased deletion frequency compared with muscle from control subjects. The frequency of the 4977-bp deletion in the worse-affected lim...

The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.

Three measures of locomotory performance and a series of variables thought to affect performance were measured in the iguanid lizard Ctenosaura similis. Burst speed is mass independent; however, endurance time at 1 km/h (EN-DUR) and maximal distance run (MAX DIS) scale as M0.3. Standard and maximal rates of O2 consumption (VO2max) scale as M0.9; VO2max averages 10-fold greater than standard metabolic rate (SMR). Three of ten enzyme activities measured exhibit significant scaling. After statistically removing the effects of body mass, multiple-regression analysis indicates that 1) 89% of the residual variation in ENDUR is correlated with variation among individuals in thigh muscle mass, VO2max, heart mass, and liver citrate synthase (CS) activity; 2) maximal CO2 consumption (VCO2max) and thigh pyruvate kinase activity statistically explain 64% of the variation in MAX DIS; 3) heart and liver masses together predict 35% of the variation in SMR; 4) thigh and liver CS activity, heart lac...

Aim-Statins decrease cardiovascular complications, but can induce myopathy. Here, we explored the implication of PGC-1α in statin-associated myotoxicity. Methods-We treated PGC-1α knockout (KO), PGC-1α over-expression (OE) and wild-type mice (WT) mice orally with 5 mg simvastatin kg -1 day -1 for 3 weeks and assessed muscle function and metabolism. Results-In WT and KO mice, but not in OE mice, simvastatin decreased grip strength, maximal running distance and vertical power assessed by ergometry. Post exercise plasma lactate concentrations were higher in WT and KO compared to OE mice. In glycolytic gastrocnemius, simvastatin decreased mitochondrial respiration, increased mitochondrial ROS production and free radical leak in WT and KO, but not in OE mice. Simvastatin increased mRNA expression of Sod1 *

± 9% vs. 105 ± 9%, p = 0.024) was also found with exercise training. Exercised mice presented with lower catalase levels (204 ± 22% vs. 141 ± 23%, p = 0.036). In a mouse model of MD, a training intervention combining aerobic and resistance exercise increased aerobic fitness and muscle strength, and mild Fiuza-Luces et al. improvements were found for activated signaling pathways involved in muscle mitochondrial biogenesis and anabolism, OXPHOS complex activity, and redox status in muscle tissue.

Most animal cells are able to meet their energy needs from the oxidation of various types of compounds: sugars, fatty acids, amino acids, but some tissues and cells of our body depend exclusively on glucose and the brain is the largest consumer of all. That is why the body has mechanisms in order to keep glucose levels stable. As it decreases, the degradation of hepatic glycogen occurs, which maintains the appropriate levels of blood glucose allowing its capture continues by those tissues, even in times of absence of food intake. But this reserve is limited, so another metabolic pathway is triggered for glucose production, which occurs in the kidneys and liver and is called gluconeogenesis, which means the synthesis of glucose from non-glucose compounds such as amino acids, lactate, and glycerol. Most stages of glycolysis use the same enzymes as glycolysis, but it makes the opposite sense and differs in three stages or also called deviations: the first is the conversion of pyruvate ...

sugars, fatty acids, amino acids, but some tissues and cells of our body depend exclusively on glucose and the brain is the largest consumer of all. That is why the body has mechanisms in order to keep glucose levels stable. As it decreases, the degradation of hepatic glycogen occurs, which maintains the appropriate levels of blood glucose allowing its capture continues by those tissues, even in times of absence of food intake. But this reserve is limited, so another metabolic pathway is triggered for glucose production, which occurs in the kidneys and liver and is called gluconeogenesis, which means the synthesis of glucose from non-glucose compounds such as amino acids, lactate, and glycerol. Most stages of glycolysis use the same enzymes as glycolysis, but it makes the opposite sense and differs in three stages or also called deviations: the first is the conversion of pyruvate to oxaloacetate and oxaloacetate to phosphoenolpyruvate. The second deviation is the conversion of fructose 1,6 biphosphate to fructose 6 phosphate and the third and last deviation is the conversion of glucose 6 phosphate to glucose.

INTRODUCTION-Ovariectomy and high fat diet (HFD) worsen obesity and metabolic dysfunction associated with low aerobic fitness. Exercise training mitigates metabolic abnormalities induced by low aerobic fitness, but whether the protective effect is maintained following ovariectomy and HFD is unknown. PURPOSE-This study determined whether, following ovariectomy and HFD, exercise training improves metabolic function in rats bred for low intrinsic aerobic capacity. METHODS-Female rats selectively bred for low (LCR) and high (HCR) intrinsic aerobic capacity (n=30) were ovariectomized, fed HFD, and randomized to either a sedentary (SED) or voluntary wheel running (EX) group. Resting energy expenditure, glucose tolerance, and spontaneous physical activity were determined midway through the experiment, while body weight, wheel running volume, and food intake were assessed throughout the study. Body composition, circulating metabolic markers, and skeletal muscle gene and protein expression was measured at sacrifice. RESULTS-EX reduced body weight and adiposity in LCR rats (-10% and -50%, respectively; P<0.05) and, unexpectedly, increased these variables in HCR rats (+7% and +37%, respectively; P<0.05) compared to their respective SED controls, likely due to dietary overcompensation. Wheel

Introduction-In the absence of exercise training, rats selectively bred for high intrinsic aerobic capacity (HCR) are protected against ovariectomy (OVX)-induced insulin resistance and obesity compared to those bred for low intrinsic aerobic capacity (LCR). Purpose-This study determined whether OVX HCR rats remain protected with exposure to high fat diet (HFD) compared to OVX LCR rats. Methods-Female HCR and LCR rats (n=36; age 27-33 weeks) underwent OVX and were randomized to a standard chow diet (NC; 5% kcal fat) or HFD (45% kcal fat), ad libitum for 11 weeks. Total energy expenditure (TEE), resting energy expenditure (REE), spontaneous physical activity (SPA), and glucose tolerance were assessed midway, while fasting circulating metabolic markers, body composition, adipose tissue distribution, and skeletal muscle AMPK and mitochondrial markers were assessed at sacrifice. Results-Both HCR and LCR experienced HFD-induced increases in total and visceral adiposity following OVX. Despite similar gains in adiposity, HCR rats were protected from HFD-induced insulin resistance and reduced TEE observed in LCR rats (P<0.05). This metabolic protection was likely attributed to a compensatory increase in SPA and associated preservation of skeletal muscle AMPK activity in HCR; whereas, HFD significantly reduced SPA and AMPK activity in LCR (P<0.05). In both lines, HFD reduced citrate synthase activity, gene expression of markers of

Swimming respirometry was employed to compare inactive metabolic rate (Rr), maximum metabolic rate (Rmax), resultant aerobic scope and maximum sustainable (critical) swimming speed (Ucrit), in growth hormone transgenic (GHT) and wild‐type (W) tilapia Oreochromis sp. hybrids. Although the Rr of GHT tilapia was significantly (58%) higher than their W conspecifics, there were no significant differences in their net aerobic scope because GHT tilapia exhibited a compensatory increase in Rmax that was equal to their net increase in Rr. As a consequence, the two groups had the same Ucrit. The GHT and W tilapia also exhibited the same capacity to regulate oxygen uptake during progressive hypoxia, despite the fact that the GHT fish were defending a higher demand for O2. The results indicate that ectopic expression of GH raises metabolic rate in tilapia, but the fish compensate for this metabolic load and preserve such physiological determinants of fitness as aerobic scope, swimming performan...

Background: Primary coenzyme Q 10 (CoQ 10 ) deficiency is rare. The encephalomyopathic form, described in few families, is characterized by exercise intolerance, recurrent myoglobinuria, developmental delay, ataxia, and seizures. Objective: To report a rare manifestation of CoQ 10 deficiency with isolated mitochondrial myopathy without central nervous system involvement. The patient was evaluated for progressive muscle weakness. Comprehensive clinical evaluation and muscle biopsy were performed for histopathologic analysis and mitochondrial DNA and respiratory chain enzyme studies. The patient began taking 150 mg/d of a CoQ 10 supplement . The elevated creatine kinase and lactate levels with abnormal urine organic acid and acylcarnitine profiles in this patient suggested a mitochondrial disorder. Skeletal muscle histochemical evaluation revealed ragged red fibers, and respiratory chain enzyme analyses showed partial reductions in complex I, I + III, and II + III activities with greater than 200% of normal citrate synthase activity. The CoQ 10 concentration in skeletal muscle was 46% of the normal reference mean. The in vitro addition of 50 µmol/L of coenzyme Q 1 to the succinate cytochrome-c reductase assay of the patient's skeletal muscle whole homogenate increased the succinate cytochrome-c reductase activity 8-fold compared with 2.8fold in the normal control homogenates. Follow-up of the patient in 6 months demonstrated significant clinical improvement with normalization of creatine kinase and lactate levels. The absence of central nervous system involvement and recurrent myoglobinuria expands the clinical phenotype of this treatable mitochondrial disorder. The complete recovery of myopathy with exogenous CoQ 10 supplementation observed in this patient highlights the importance of early identification and treatment of this genetic disorder.

Objectives: Adenomyosis is a benign uterine disease that occurs with the invasion of the endometrial gland and stoma into the myometrium. The etiology and molecular pathology of adenomyosis are not yet fully understood. Tissue samples of patients diagnosed with adenomyosis and healthy endometrial tissues were investigated for the lipogenesis and cholesterol synthesis pathways. It was aimed to determine the difference between adenomyosis and healthy endometrial tissues in terms of lipid metabolism and to investigate the mechanism of adenomyosis in this context. Methods: Formalin-fixed paraffin-embedded archival tissues were used in the current retrospective study. A total of 76 patient samples and 3 groups were used. Group 1: adenomyotic tissue (n=28), Group 2: eutopic endometrial tissue (n=30), and Control Group (n=18). In these groups, Sterol regulatory element binding protein 1 (SREBP1) molecule, fatty acid synthase (FASN), acetyl-CoA carboxylase (ACACA), ATP-citrate lyase (ACLY), HMG-CoA reductase (HMGCR), and HMG-CoA synthase (HMGCS) markers were evaluated by using RT-PCR method. Results: Statistically significant differences (p<0.05) were found between the groups regarding expression levels of HMGCR, HMGCS, ACLY, ACACA, and SREBP1. HMGCR, HMGCS, ACLY, and SREBP1 gene expression levels between Group 1 and Group 2 and HMGCS, ACACA, ACLY, and SREBP1 gene expression levels between Group 1 and Control Group were determined as statistically different. A significant difference was detected only in HMGCR gene expression levels between Group 2 and the Control Group. Conclusions: These results show that genes involved in lipid metabolism may be associated with the molecular pathogenesis of adenomyosis.

Hypersaline environments are widely distributed on Earth. They are of various forms, including natural permanent saline lakes and salt marshes. They have also been created artificially, due primarily to anthropogenic activities and exist as solar salterns . Hypersaline environments can be classified as thalassohaline and athalassohaline. The former were created by the evaporation of seawater. Sodium and chloride ions dominate and the pH is nearly neutral. Athalassohaline environments have a higher concentration of divalent cations than the monovalent ions and a low pH .

Prior studies with carbonic anhydrase (CA) inhibitors implicated mitochondrial CA in ureagenesis and gluconeogenesis. Subsequent studies identified two mitochondrial CAs. To distinguish the contribution of each enzyme, we studied the effects of targeted disruption of the murine CA genes, called Car5A and Car5B . The Car5A mutation had several deleterious consequences. Car5A null mice were smaller than wild-type littermates and bred poorly. However, on sodium–potassium citrate-supplemented water, they produced offspring in expected numbers. Their blood ammonia concentrations were markedly elevated, but their fasting blood sugars were normal. By contrast, Car5B null mice showed normal growth and normal blood ammonia levels. They too had normal fasting blood sugars. Car5A / B double-knockout (DKO) mice showed additional abnormalities. Impaired growth was more severe than for Car5A null mice. Hyperammonemia was even greater as well. Although fertile, DKO animals were produced in less-th...

as those occurring upon substrate binding or in different Biologie Physico-Chimique, 13 crystal forms of the same protein. Using, as sole information, the atomic coordinates of a pair of protein structures, the procedure first generates rue Pierre et Marie Curie Paris, 75005, France structure alignments, which optimize the root-mean-square deviation of the backbone atoms. To this end, equivalent secondary structures and/or 2 Unite ´de Conformation des loops from both proteins are combined by a multiple linkage hierarchic Macromole ´cules Biologiques clustering algorithm, which generates several intertwined clustering trees. Universite ´Libre de Bruxelles Automatic analysis of these clustering trees is used to dissect the CP 160/16, Avenue P. He ´ger mechanism of the conformational change. It allows the identification of the 1050 Bruxelles, Belgium static core, representing the collection of secondary structures which undergo no structural changes, as well as other entities which move like rigid bodies. It also permits the description of the movement of secondary structures or loops relative to this core or entities. Using this information, it can be inferred whether a particular conformational change involves shear or hinge motion, or components of both. The algorithm is applied to the analysis of the conformational changes of citrate synthase, lactate dehydrogenase, lactoferrin and b-glucosyltransferase, representing typical examples of shear-and hinge-type mechanisms, and a varied range in movement size. The results are shown to be in excellent agreement with previous analyses, and to provide additional information which gives a more complete and objective picture of the conformational change. Using our automatic algorithm, we find that any conformational change may be viewed as having components of both shear-and hinge-type motion. Determining which of these is most appropriate requires the combination of the information provided by our procedure with detailed knowledge of the protein tertiary structures.

Human mitochondrial disease exhibits large variation of clinical phenotypes, even in patients with the same causative gene defect. We illustrate this heterogeneity by confronting clinical and biochemical data of two patients with the uncommon pathogenic homoplasmic NC_012920.1(MT-ATP6):m.9035T&gt;C variant in MT-ATP6. Patient 1 presented as a toddler with severe motor and speech delay and spastic ataxia without extra-neurologic involvement. Patient 2 presented in adolescence with ataxia and ophthalmoplegia without cognitive or motor impairment. Respiratory chain complex activities were normal in cultured skin fibroblasts from both patients when calculated as ratios over citrate synthase activity. Native gels found presence of subcomplexes of complex V in fibroblast and/or skeletal muscle. Bioenergetic measurements in fibroblasts from both patients detected reduced spare respiratory capacities and altered extracellular acidification rates, revealing a switch from mitochondrial respir...

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-

The Baetis vernus group (Ephemeroptera: Baetidae) – which includes B. brunneicolor McDunnough, B. bundyae Lehmkuhl, B. hudsonicus Ide, B. jaervii Savolainen, B. liebenauae Keffermuller, B. macani Kimmins, B. subalpinus Bengtsson, B. tracheatus Keffermuller & Machel, and B. vernus Curtis – is both diverse and taxonomically tangled. Some members of the group – B. brunneicolor , B. bundyae , and B. hudsonicus – have been previously found in North America. The remainder of the group is known to be only of Palearctic distribution, including B. vernus , which has a wide trans-Palearctic distribution. We report the collection of specimens from the Northwest Territories and British Columbia that we have identified as B. vernus using DNA barcoding and morphological examination and provide characters to assist separation of the North American members of the group from B. vernus . A genetically cohesive Holarctic clade for B. vernus likely relates to a Beringian dispersal event. This substanti...

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed different...

SUMMARY We measured activity levels, oxygen consumption, metabolic enzyme activity,breathing frequency, heart rate and blood chemistry variables of juvenile green turtles exposed to a laboratory simulation of subtropical winter and summer temperatures (17-26°C) and photoperiod (10.25 h:13.75 h to 14 h:10 h light:dark). The activity level of turtles had a significant effect on oxygen consumption and breathing frequency but there was no significant change in activity level between the summer and winter simulations. There was a moderate 24-27% decrease in oxygen consumption during exposure to winter conditions compared with summer conditions, but this difference was not statistically significant. Likewise, there was no statistically significant difference in breathing frequency between summer and winter simulations. Exposure to winter conditions did result in a significant decrease in activity of the aerobic enzyme citrate synthase. By contrast, activities of the glycolytic enzymes pyr...

Common terns (Sterna hirundo), sooty terns (S. fuscata) and brown noddies (Anous stolidus) are phylogenetically related seabirds that differ in field activity levels and daily energy expenditure. To test whether muscle metabolic capacities co-evolve with activity levels and energy expenditure, we collected pectoral muscle biopsies from members of each species, and measured the activities of key enzymes in oxidative metabolism (citrate synthase, CS), anaerobic metabolism (lactate dehydrogenase, LDH), glycolysis (pyruvate kinase, PK), fatty acid oxidation (3-hydroxyacyl CoA dehydrogenase) and phosphocreatine hydrolysis (creatine phosphokinase, CPK). We hypothesized that temperate-breeding common terns would have higher enzyme activities than the two tropical species (sooty terns and brown noddies); consistent with the higher activity level of common terns. There were no differences in enzyme activities among adults of the three species. Common tern chicks within 2-3 days of flight had two-fold higher pyruvate kinase activity than adults, suggesting an increased glycolytic capacity in the chicks. Given the lack of difference among species at the enzymatic level, our results support the notion that behavior and whole organism performance can evolve considerably before there are detectable changes in underlying lower-level physiological/ biochemical traits.

The energy metabolism was evaluated in gastrocnemius muscle from 3-month-old rats subjected to either mild or severe 4-week intermittent normobaric hypoxia. Furthermore, 4-week treatment with CNS-acting drugs, namely, c~-adrenergic (g-yohimbine), vasodilator (papaverine, pinacidil), or oxygen-increasing (almitrine) agents was performed. The muscular concentration of the following metabolites was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactateto-pyruvate ratio; citrate, c~-ketoglutarate, succinate, malate; aspartate, glutamate, alanfl~e; ammonia; ATP, ADP, AMP, creatine phosphate. Furthermore the Vmax of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The adaptation to chronic intermittent normobaric mild or severe hypoxia induced alterations of the components in the anaerobic glycolytic pathway [as supported by the increased activity of lactate dehydrogenase and/or hexokinase, resulting in the decreased glycolytic substrate concentration consistent with the increased lactate production and lactate-to-pyruvate ratio] and in the mitochondrial mechanism [as supported by the decreased activity of malate dehydrogenase and/or citrate synthase resulting in the decreased concentration of some key components in the tricarboxylic acid cycle]. The effect of the concomitant pharmacological treatment suggests that the action of CNS-acting drugs could be also related to their direct influence on the muscular biochemical mechanisms linked to energy transduction.

RESUMO -Foi determinada a atividade das enzimas NADH desidiogenase, NADH citocromo e redutase, succinato desidiogenase, succinato citocromo e redutase, citocromo e oxidase e citrato sintase em mitocôndrias de músculo esquelético humano normal e doente (suspeito de miopatia mitocondrial). O grupo controle foi constituído de 13 indivíduos normais e que não faziam uso contínuo de fárrnacos. O grupo doente era constituído de 10 pacientes cujo diagnóstico anatomopatológico indicava suspeita de miopatia mitocondrial. Observou-se redução na atividade das enzimas em todos os pacientes: 7 com anormalidades em todas as enzimas ensaiadas; 2 com deficiências em todas as enzimas exceto na citocromo e oxidase; e 1 paciente com disfunção apenas na atividade da succinato desidiogenase e succinato citocromo e redutase. Este perfil possibilitou caracterizar múltiplas deficiências ou deficiência combinada da cadeia respiratória, além da disfunção na citrato sintase em 9 pacientes. Um dos casos constituiu exceção, sendo a deficiência enzimática restrita ao complexo II. Foi possível concluir que a metodologia usada é adequada e facilmente aplicável aos objetivos clínicos. Os resultados obtidos possibilitam a caracterização dos complexos enzimáticos mitocondriais deficientes, mostrando que tais enfermidades são originadas de disfunção no metabolismo energético. PALAVRAS-CHAVE: mitocôndria, músculo esquelético humano, miopatia mitocondrial, NADH desidiogenase, NADH citocromo e redutase, succinato desidrogenase. succinato citocromo e redutase, citocromo e oxidase, citrato sintase. ABSTRACT-The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome e oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.

The effect of administration of fish oil by gavage on key enzyme activities of glucose metabolism of the thymus, spleen, and mesenteric lymph nodes was investigated. 2. The activities of hexokinase, glucose-6-phosphate dehydrogenase, and citrate synthase in the lymphoid organs were markedly raised due to a daily administration of fish oil by gavage (0.4% of body weight). 3. These findings indicate that the therapeutic utilization of fish oil does affect the metabolism of the lymphoid organs, and possibly immune function; however, the mechanism involved remains to be investigated. GEN PHARMAC 27;6:991-994, 1996.

transaminase (GOT1), which catalyzes the reversible transformation of oxaloacetate and glutamate to aspartate and α-ketoglutarate. Thus, the increase of the co-substrate (oxaloacetate) shifts the equilibrium of the reaction to the right side, thereby decreasing the blood glutamate concentration and lowering the brain glutamate levels (see [3,4] for review). Based on this mechanism, we have demonstrated that a decrease of blood glutamate levels, by means of an intravenous (i.v.) administration of oxaloacetate, leads to a larger natural glutamate gradient from the brain to the blood, which facilitates the decreases of extracellular levels of brain glutamate inducing a neuroprotective effect after ischemic insults [5,6]. Therefore, the administration of oxaloacetate may represent an interesting strategy to reduce the neurological complications associated to ECC, although further experimental studies are necessary to elucidate this hypothesis.

Four species of planktonic calanoid copepods that co-occur in the California Current System (Eucalanus californicus Johnson, Rhincalanus nasutus Giesbrecht, Calanus pacificus californicus Brodsky, and Metridia pacifica Brodsky) were investigated for evidence of seasonal dormancy in the San Diego Trough. Indices used to differentiate actively growing from dormant animals included developmental stage structure and vertical distribution; activity of aerobic metabolic enzymes (Citrate Synthase and the Electron Transfer System complex); investment in depot lipids (wax esters and triacylglycerols); in situ grazing activity from gut fluorescence; and egg production rates in simulated in situ conditions. None of the 4 species exhibited a canonical calanoid pattern of winter dormancyi.e., synchronous developmental arrest as copepodid stage V, descent into deep waters, reduced metabolism, and lack of winter reproduction. Instead, Calanus pacificus californicus has a biphasic life history in this region, with an actively reproducing segment of the population in surface waters overlying a deep dormant segment in winter. Eucalanus californicus is dormant as both adult females and copepodid V's, although winter females respond relatively rapidly to elevated food and temperature conditions; they begin feeding and producing eggs within 2-3 days. Rhincalanus nasutus appears to enter dormancy as adult females, although the evidence is equivocal. Metridia pacifica shows no evidence of dormancy, with sustained active feeding, diel vertical migration behavior, and elevated activity of metabolic enzymes in December as well as in June. The four species also differ markedly in water content, classes of storage lipids, and specific activity of Citrate Synthase. These results suggest that copepod dormancy traits and structural composition reflect diverse adaptations to regional environmental conditions rather than a uniform, canonical series of traits that remain invariant among taxa and fixed across a species' range. Such

The Galápagos marine iguana, Amblyrhynchus cristatus, is unique among lizards in foraging subtidally, leading to activity across a broad range of ambient temperatures (∼ 14-40 °C). To determine whether the marine iguana shows any biochemical changes consistent with maintaining enzyme function at both warm and cold body temperatures, we examined the function of the aerobic enzyme citrate synthase (CS) and the muscle isoform of the anaerobic enzyme lactate dehydrogenase (A 4 -LDH) in A. cristatus and a confamilial species, Iguana iguana, from 14 to 46 °C. We also deduced amino acid sequences from cDNA of each enzyme. In CS, despite two amino acid substitutions, we found no difference in the apparent Michaelis-Menten constant K m of oxaloacetate at any temperature, indicating that the substrate affinity of CS in A. cristatus has not adapted to changes in thermal environment. In A 4 -LDH, we used site-directed mutagenesis to show that the substitutions T9A and I283V (A. cristatus → I. iguana) individually have no effect on kinetics, but together significantly decrease the K m of pyruvate and catalytic rate constant (k cat ) of the A. cristatus ortholog. Thus, our data show that A. cristatus A 4 -LDH has not become cold adapted in response to this species' aquatic foraging behavior, and instead may be consistent with moderate warm adaptation with respect to the I. iguana ortholog.

In most tissues the mitochondrial ATP-synthase plays a central role by synthesizing the bulk of ATP. According to the classical theory of respiratory control, flux through this enzyme is solely determined by substrate (ADP) concentration while the enzyme has a fixed capacity. However, in different cell types such as rat cardiomyocytes and neurons, dog heart, human fibroblasts, and skeletal and heart muscle from children, it has been shown that active regulation of the mitochondrial ATP-synthase in response to cellular energy demand exists. For example, in rat cardiomyocytes the mitochondrial ATP-synthase activity is down-regulated in response to anoxia or mitochondrial uncoupling. By this mechanism cellular ATP is conserved, as under these conditions the ATP-synthase would work in reverse and hydrolyze ATP. When cardiomyocytes are stimulated to contract, ATP-synthase activity is up-regulated in line with the increased energy demand. Preincubation of the cardiomyocytes with positive inotropic substances results in further up-regulation of the ATP-synthase. By blocking calcium transport, it has been shown that the up-regulation of the enzyme is calcium-dependent. On a molecular level, up-regulation is probably mediated by the calcium-binding inhibitor protein (CaBI) and down-regulation via the inhibitor protein IF 1 . The ATP-synthase system is disturbed under several pathophysiological conditions. First, mutations can cause a primary defect in the mitochondrial ATP-synthase (respiratory chain defect). Furthermore, secondary defects of the ATP-synthase occur. In rat models abnormalities of ATP-synthase can be detected in different types of cardiomyopathy/heart hypertrophy. The changes are reversible in response to treatment of the heart diseases. Abnormalities of the ATPsynthase system can be observed in fibroblasts from patients with neuronal ceroidlipofuscinoses. Toxic metabolites accumulating in methylmalonic acidurias can inhibit ATP-synthase. When neurons are incubated with 3-OH glutarate-a substance accumulating in glutaric aciduria I-as a model for glutaric aciduria I, ATP-synthase activity is compromised. This lack of energy may lead to Ôslow onsetÕ excitotoxicity and finally cell death. Cells can be rescued by adding creatine to the incubation medium. In D D -2-hydroxyglutaric aciduria, inhibition of the ATP-synthase has been observed.

In previous studies regulation of the FIFO-ATPase of mitochondrial complex V (ATP synthase) has been demonstrated in rat cardiomyocytes, canine mycocardium and skeletal muscle from children. The aim of the present study was to examine regulation of ATP synthase in human myocardium in response to different metabolic states. 2. Biopsy material was obtained from 10 children undergoing cardiac surgery. Mitochondria in the postnuclear supernatant were incubated under different metabolic conditions for 15min and then broken by sonication. ATP synthase was measured spectrophotometrically using a coupled enzyme assay. 3. ATP synthase can be rapidly measured in sonicated preparations of heart mitochondria from children. We show that direct regulation at the level of ATP synthase occurs in these mitochondria. ATP synthase capacity is decreased in response to blocking of the respiratory chain by cyanide (mimicking anoxia) or uncoupling of mitochondria, falling to 76 % and 66 % of control values respectively. Upregulation of ATP synthase can be demonstrated in heart mitochondria when the calcium concentration in the incubation medium is increased to 5 pM (130% of control). 4. ATP synthase is actively regulated in heart mitochondria from children. The enzyme is upregulated in response to increased calcium. This transition may reflect the increased energy demand when cardiac workload is increased.

Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115-1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the "malate-like intermediate" found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108-1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y. O. Belikova, et al., Biochim. Biophys. Acta 936 1-9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.

To assess the potential adaptive value of mtDNA, we evaluated functional properties and thermal sensitivity of key mitochondrial enzymes in two species that have originally evolved in different thermal environments (arctic charr, Salvelinus alpinus, and brook charr, S. fontinalis), as well as in their hybrids. We measured the activity of two enzymes of the electron transport system (cytochrome c oxidase and NADH-ubiquinone oxidoreductase), one enzyme of the mitochondrial matrix (citrate synthase), and one enzyme of the anaerobic glycolysis (lactate dehydrogenase) in the red muscle at three temperatures (61C, 121C and 181C). Surprisingly, the species presented no significant differences in enzyme activity, thermal sensitivity or thermostability of key metabolic enzymes even though they evolved in different thermal environments and present important differences in amino acid sequences. It seems that amino acid substitutions between those species have minor impact on the functional properties of mitochondrial enzymes studied. The thermal sensitivity results (Q 10 ) obtained for inner-membrane mitochondrial enzymes differed somewhat from those of mitochondrial matrix or cytosolic enzymes. This result indicates the modulation of thermal sensitivity of all mitochondrial inner-membrane processes by a common parameter, which could be the structural and functional properties of membrane phospholipids.

The goal of the present study was to discern the cellular mechanism(s) that contributes to the age-associated decrease in skeletal muscle aerobic capacity. Skeletal muscle mitochondrial content, a parameter of oxidative capacity, was significantly lower (25 and 20% calculated on the basis of citrate synthase and succinate dehydrogenase activities, respectively) in 24-mo-old Fischer 344 rats compared with 6-mo-old adult rats. Mitochondria isolated from skeletal muscle of both age groups had identical state 3 (ADP-stimulated) and ADP-stimulated maximal respiratory rates and phosphorylation potential (ADP-to-O ratios) with both nonlipid and lipid substrates. In contrast, mitochondria from 24-mo-old rats displayed significantly lower state 4 (ADP-limited) respiratory rates and, consequently, higher respiratory control ratios. Consistent with the tighter coupling, there was a 68% reduction in uncoupling protein-3 (UCP-3) abundance in mitochondria from elderly compared with adult rats. Co...

Background-Although a number of exercise systems have been developed to mitigate the physiological deconditioning that occurs in microgravity, few have the capacity to positively impact multiple physiological systems and still meet the volume/mass requirements needed for missions beyond low earth orbit. The purpose of this study was to test the gravity-independent Multi-Mode Exercise Device (M-MED) for both resistance (RE) and aerobic (AE) training stimuli. Methods-Eight men and nine women (mean age 22.0±0.4 years) completed five weeks of training on the M-MED: RE 4×7 squats two days a week, and AE 4×4-min rowing bouts at ~90% VO 2 max three days a week. Pre-and post-training data collection included an aerobic capacity test, MR imaging, strength testing, and vastus lateralis muscle biopsy. Results-VO 2 max increased 8%, 3RM strength 18%, and quadriceps femoris cross-sectional area (CSA) 10%. Knee extensor strength increased at all isokinetic speeds tested. Subjects also demonstrated improved resistance to fatigue in knee extension. At the cellular and molecular level, the biopsy revealed increases in mixed myofiber CSA (13%), citrate synthase activity (26%), total RNA concentration (24%), IGF-I mRNA (77%), Type IIa Myosin Heavy Chain (MHC) mRNA (8%), and concomitant decrease in Type IIx MHC mRNA (-23%). None of the changes were gender-specific.

Two common bean (Phaseolus vulgaris L.) genotypes differing in aluminum (Al) resistance, Quimbaya (Al-resistant) and VAX-1 (Al-sensitive) were grown in hydroponics for up to 25 h with or without Al, and several parameters related to the exudation of organic acids anions from the root apex were investigated. Al treatment enhanced the exudation of citrate from the root tips of both genotypes. However, its dynamic offers the most consistent relationship between Al-induced inhibition of root elongation and Al accumulation in and exclusion from the root apices. Initially, in both genotypes the short-term (4 h) Al-injury period was characterized by the absence of citrate efflux independent of the citrate content of the root apices, and reduction of cytosolic turnover of citrate conferred by a reduced Nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase (EC 1.1.1.42) activity. Transient recovery from initial Al stress (4-12 h) was found to be dependent mainly on the capacity to utilize internal citrate pools (Al-resistant genotype Quimbaya) or enhanced citrate synthesis [increased activities of NAD-malate dehydrogenase (EC 1.1.1.37) and ATP-phosphofructokinase (EC 2.7.1.11) in Al-sensitive VAX-1]. Sustained recovery from Al stress through citrate exudation in genotype Quimbaya after 24 h Al treatment relied on restoring the internal citrate pool and the constitutive high activity of citrate synthase (CS) (EC 4.1.3.7) fuelled by high phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity. In the Alsensitive genotype VAX-1 the citrate exudation and thus Al exclusion and root elongation could not be maintained coinciding with an exhaustion of the internal citrate pool and decreased CS activity.