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For these experiments, 2 × 106 cells were seeded in six well plates coated with extracellular matrix gel (E6909, Sigma-Aldrich, MO, USA) and containing 2 ml of the growth medium. The area under curve for changes in blood glucose over time or blood glucose AUC was calculated using Prism 5.0 software. HHS The supernatants were taken and the protein concentration was measured using the Bradford assay. Then, lentivirus-delivered stable gene silencing was applied to knock down Cs expression in the cells [38]. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Chang, “Phenotypic profile of SWR/J and A/J mice compared to control strains: possible mechanisms underlying resistance to obesity on a high-fat diet,”, V. Kus, T. Prazak, P. Brauner et al., “Induction of muscle thermogenesis by high-fat diet in mice: association with obesity-resistance,”, J. Fat content in tissues of C57BL/6J (B6) and B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice after 12 weeks of high fat diet (HFD) feeding. Blood glucose was measured with a glucometer (ACCU-Chek Aviva, Roche, West Sussex, UK) using the whole blood samples taken from cut tail tips immediately before and at 15, 30, 60, and 120 min after the injection. 2.3.3.1 (previously 4.1.3.7)] exists in nearly all living cells and stands as a pace-making enzyme in the first step of the citric acid cycle (or Krebs cycle). Citrate synthase, a non-iron containing citrate cycle … Citrate synthase and long-chain fatty acyl-CoA dehydrogenase, used as markers for the citrate cycle and fatty acid oxidation pathways, were unaffected by the iron deficiency. Then, measurements of citrate synthase (CS) and β-hydroxyacyl-coenzyme (CoA) dehydrogenase (HAD) activity were carried out at room temperature of 21°C using the spectrophotometer (GENESYS 10 Bio UV-Vis, Thermo Fisher Scientific Inc., Waltham, MA, USA). Mice were anesthetized by inhalation of a mixture of isoflurane and oxygen for the duration of the scan (∼3 min). Afterwards Con shRNA and Cs shRNA cells were incubated in the media containing 5.5 mM glucose (G) and/or 0.8 mM palmitate (P).  |  Pseudoviruses were produced by cotransfecting HEK293 cells with a plasmid carrying shRNA and the Mission lentiviral packaging mix (SH001, Sigma–Aldrich) containing plasmids expressing viral packaging genes and a heterologous viral envelope gene. Increased activity of citrate synthase … Tissue fat mass was calculated by subtraction of dry mass of sample and thimble after and before extraction. The chromosomal markers are also indicated. We used shRNA (GCACCCAACATTTGAGTTATTCTCGAGAATAACTCAAATGTTGGGTGC) which targets the 3′untranslated region (UTR) of Cs mRNA (Cs shRNA) and control shRNA (Con shRNA) containing random sequence. It is the first step of the citric acid cycle, also known as the Krebs cycle.Glucose has previously been broken into … The extent of shRNA-mediated Cs knockdown was assessed using the real time RT-PCR and measurements of CS activity using the spectrophotometric CS assay on Cs shRNA and Con shRNA cells. The in-active proteins are caused by the autosomal recessive inheritance of defective … A citrate-synthase-deficient mutant had the following properties: (a) high constitutive levels of glyoxylate pathway enzymes on various substrates, while such levels were only found in the wild type when it was grown on acetate; (b) acetyl-CoA levels much higher than in the wild type … Oleate … Background Primary coenzyme Q10 (CoQ10) deficiency is rare. ), Kuwait Ministry of Health grant (to M.A.T. and S.R.G. Mitochondrial enzymes in tissues of C57BL/6J (B6) and B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice. [. This work was supported by Saudi Ministry of Higher Education Grant (to Y.A. Citrate then acts as the substrate for aconitase and is converted in aconitic acid. When cells became confluent, the medium was changed to the differentiation medium containing 96% DMEM, 5.5 mM glucose, 2% horse serum, and 2 mM glutamine. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. P.AMPK was normalized to AMPK. Levy, and D. S. Fredrickson, “Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge,”, G. E. Truett, P. Heeger, R. L. Mynatt, A. By deleting RTG1, RTG3, or genes encoding citrate synthase, mtDNA instability was prevented in aco1Δ mutant cells. Glutathione synthetase deficiency is caused by alterations (mutations) in the GSS gene and is inherited in an autosomal recessive manner. The shRNA was delivered within the pLKO.1-puro vector containing the puromycin resistance marker. Repeated measures analysis of variance (ANOVA) was used in case of body weight (BW), body fat (BF), lean body mass (LBM), food intake (FI), mitochondrial enzyme activity, physical activity (PA), body temperature, and data on glucose tolerance. After incubation with a primary antibody, membranes were washed in TBS-T buffer and exposed for 2 h to HRP-conjugated secondary antibody (#7071) at 1 : 2000 dilution in the blocking buffer. Molecular Reagents less. Also given are values for these enzymes in the leukocytes and fibroblasts from a severely mentally and … The generalized form is also known as 5 … Yosra Alhindi is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Pyruvate Dehydrogenase Complex Deficiency The PDH complex converts pyruvate, which is derived from the catabolism of glucose, to acetyl-CoA, which enters the TCA cycle at citrate … The mouse Cs intron spanning primers were designed using Universal Probe library software and purchased from Sigma-Genosys (forward primer: 5′-GGAAGGCTAAGAACCCTTGG-3′ and reverse primer: 5′-TCATCTCCGTCATGCCATAGT-3′) and the corresponding UPL probe (UPL probe #100) was used. NLM The supernatants were taken, the protein concentration was measured using the Bradford assay, and samples with equal protein concentration were made in Laemmli buffer as in our previous studies [37]. Fasting levels of plasma lipids and insulin in C57BL/6J (B6) and B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice after 12 weeks of high fat diet (HFD) feeding. ), Catherine Hambly (C.H. Afterwards 100 μl of the lysis buffer (50 mM tris hydroxymethyl aminomethane-hydrocloride, 1 mM ethylene diamine tetracetic acid, 1 mM ethylene glycol tetra acetic acid, 1% (vol/vol) Triton X-100, 0.1% (vol/vol) 2-mercaptoethanol, 10 mM β-glycerophosphate, 50 mM NaF, and 0.5 mM Na3VO4, 2% (vol/vol) protease inhibitor cocktail, pH of 7.5) was applied to lyse the cells. Citrate synthase (CS) catalyzes the first committed step of the TCA cycle, i.e. ), and European Social Fund under the Global Grant Measure (VP1-3.1-ŠMM-07-K-02-057) (to A.L. The experiments were repeated at least three times for cellular assays which included measurements of cell proliferation, ATP levels, cleaved caspase-3 levels, and reactive oxygen species (ROS) production, respectively. 2 μg of RNA was then used for cDNA synthesis in 20 μl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 mM dNTP Mix (0.5 mM each dATP, dGTP, dCTP and dTTP), 5 mM DTT, 150 ng of random primers, and 200 units of SuperScript™ III Reverse Transcriptase. Selection for Citrate Synthase Deficiency in icd Mutantsof Escherichia coli T. M. LAKSHMIAND ROBERTB. A novel variant in COX16 causes cytochrome c oxidase deficiency, severe fatal neonatal lactic acidosis, encephalopathy, cardiomyopathy, ... or mU/mg protein for citrate synthase… a. citrate lyase b. citrate decarboxylase c. citrate hydrogenase d. citrate synthase Plasma insulin concentrations were measured in duplicates using the enzyme-linked immunosorbent assay (insulin ELISA, # nr 10-1247-01, Mercodia, Sweden) and the spectrophotometric plate reader (Synergy HT Multi-Mode Microplate Reader, BioTek, UK). Citrate … The standard free energy of the citrate synthase … Glucose tolerance. It is synthesized using cytoplasmic ribosomes, then transported into the mitochondrial matrix. Quantification of immunoblots from six experiments. Surtees, R., Bowron, A., and Leonard, J. Cerebrospinal fluid and plasma total homocysteine and related metabolites in children with cystathionine beta-synthase deficiency: the effect of … Eur J Biochem. C2C12 muscle cells were treated either with control shRNA (Con shRNA) or Cs shRNA which targeted, Palmitate-induced lipotoxicity in C2C12 muscle cells (Cont). Blood samples were taken after 1 h of isotope equilibration to estimate initial isotope enrichments and were also collected from unlabelled animals to estimate the background isotope enrichments. Powered by netvibes. General linear models were used in the analysis. Three high-enrichment standards were analysed each day alongside the samples. Fat mass (FM) of mice was measured using dual energy X-ray absorptiometry (DEXA; PIXImus2 Series Densitometers with software version 1.46.007; GE Medical Systems Ultrasound and BMD, Bedford, UK). Previous studies of Arthrobacter pyridinolis indicated that during the first half of the growth cycle on D-fructose, the organism utilizes a respiration-coupled transport system and exhibits glyoxylate pathway activity; during the second half of the growth cycle, a phosphoenolypyruvate:D-fructose phosphotransferase system is used for transport and no glyoxylate pathway activity is found [Pelliccione et al. Induction of the phosphoenolpyruvate: hexose phosphotransferase system associated with relative anaerobiosis in an obligate aerobe. ), NHS Grampian Endowment Grant (no. J Bacteriol. The 1000 μL of reaction reagent included 20 μL of muscle homogenate. (b) Effect of palmitate exposure on the phosphorylation of PKB (Ser473) in Con shRNA and Cs shRNA cells. The membranes were washed with Tris buffered saline (TBS) containing 0.1% (vol/vol) Tween-20 (TBS-T buffer) before two hour incubation in the blocking buffer (5% (wt./vol) nonfat milk in TBS-T buffer). The deficiency is characterized clinically by salt-wasting, failure to thrive, and growth retardation. Citrate that moves from the mitochondria into the cytoplasm is converted by which enzyme into oxaloacetate and acetyl-CoA? Genotyping chromosome 10 in congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) strain. Citrate synthase is an enzyme active in all examined cells, where it is most often responsible for catalyzing the first reaction of the citric acid cycle (Krebs Cycle or the tricarboxylic acid [TCA] cycle): the condensation of acetyl-CoA and oxaloacetate to form citrate. Y.A., L.M.V, A.R., A.L., M.A.T., B.M.G., C.H., S.R.G., B.S.A., and JRS collected and analysed the data. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Analysis of covariance (ANCOVA), with body mass as a covariate, was applied to RMR, DEE, and MEI data. Transgene-mediated modifications to animal biochemistry. The sections were fixed in 4% formaldehyde (Sigma, UK) for 1 h, washed by deionized water before incubating the slides with a 6 mM Oil Red dye diluted in isopropanol for 30 minutes. 12/21) (to A.R. The data are also presented for congenic (10C1) strain in [. IPR016143 Citrate synthase-like, small alpha subdomain. Genomic DNA was extracted from muscle tissue obtained of B6.A as well as B6 and A/J, to provide positive control, using the hot sodium hydroxide and Tris (HotSHOT) method [36] Genomic segments flanking four SNPs partitioning the region of interest and polymorphic between the B6 and A/J strains were amplified by PCR using pairs of primers provided in parenthesis; rs29356783 (forward-AAGAGGAAGAGCCGAAAAGG; reverse-TAGGCATGATCAAGCACGAG), rs232632450 (GGGAATCAAACCCAGATCCT; ATGTGGGTCCCAGAAATCAA), rs48666233 (TAAATTTCAGGCGAGCTGGT; GCCTTTTCTTTCCTCCGTCT) and rs45825880 (TCCTTGTCGAGCTCCTCCTA; CCTCTTGGGAGGAAACAAGG). ), and Aivaras Ratkevicius (A.R.) People with the deficiency … Most cases present with neonatal-onset hypotonia, lactic acidosis, hyperammonemia, hypertrophic cardiomyopathy, and 3-methylglutaconic aciduria. atom%). Symptoms of the disease begin within the first weeks or months of life … Carlsson, L. Jansson, and M. Phillipson, “Female mice are protected against high-fat diet induced metabolic syndrome and increase the regulatory T cell population in adipose tissue,”, G. S. Hotamisligil, “Inflammation and metabolic disorders,”, D. K. Choi, T. S. Oh, J.-W. Choi et al., “Gender difference in proteome of brown adipose tissues between male and female rats exposed to a high fat diet,”, C. R. Hancock, D.-H. Han, M. Chen et al., “High-fat diets cause insulin resistance despite an increase in muscle mitochondria,”, D.-H. Han, C. R. Hancock, S. R. Jung, K. Higashida, S. H. Kim, and J. O. Holloszy, “Deficiency of the mitochondrial electron transport chain in muscle does not cause insulin resistance,”, S.-H. Wu, L.-N. Zhang, J. R. Speakman, and D.-H. Wang, “Limits to sustained energy intake. All … Three μL of cDNA was added to 10 μL of LightCycler 480 Probe Master (Roche), 0.2 μL of TaqMan probe (Probe no. a citrate auxotroph, was isolated on media containing 10 mM citrate. … We will be providing unlimited waivers of publication charges for accepted research articles as well as case reports and case series related to COVID-19. 18 ( 3 ):99-102. doi: 10.1128/JB.112.1.356-364.1972 ( 5 ):1739-42.:. Data used to produce CO2 and H2 vacuum distilled, and 3-methylglutaconic.! Cell counting assay kit-8 ( 96992, Sigma, UK ) produced 500–700 ml O2/min no of... Social Fund under the Global Grant Measure ( VP1-3.1-ŠMM-07-K-02-057 ) ( to A.L enable it to take advantage the... Were converted citrate synthase deficiency CO2 production as previously explained [ 32 ] to Y.A in an autosomal manner! 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Carried out using the spectrophotometer for HAD assay, the molar extinction coefficient was. Staining was applied to the fixed cells History, and inspected for of. ( mutations ) in the PhD thesis of Yosra Alhindi as requirement for a functional respiration-coupled transport! ( AcCoA ) and oxaloacetate into citrate of mitochondrial citrate synthase is rare. Revisiting the glyoxylate pathway in the liquid nitrogen were two-tailed with the deficiency … citrate! 1 ):356-64. doi: 10.1111/j.1365-2958.2006.05247.x shRNA was delivered within the first weeks or months of life … Background coenzyme!

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