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Sample records for acetyl coa carboxylase

  1. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    International Nuclear Information System (INIS)

    Roman-Lopez, C.R.; Allred, J.B.

    1986-01-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind [ 14 C]methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase

  2. Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

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    Jang Eun Lee

    Full Text Available Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2 regulates fatty acid oxidation (FAO by inhibiting carnitine palmitoyltransferase 1 (CPT1, a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

  3. Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding

    International Nuclear Information System (INIS)

    Roman-Lopez, C.R.; Goodson, J.; Allred, J.B.

    1987-01-01

    Conditions are described under which monomeric [ 14 C]methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of the [ 14 C]methyl avidin was known, the quantity of avidin bound and therefore the quantity of biotin enzyme could be calculated. The method is illustrated by the analysis of purified acetyl CoA carboxylase and is applied to the analysis of biotin enzymes in isolated rat liver mitochondria

  4. Biotin augments acetyl CoA carboxylase 2 gene expression in the hypothalamus, leading to the suppression of food intake in mice.

    Science.gov (United States)

    Sone, Hideyuki; Kamiyama, Shin; Higuchi, Mutsumi; Fujino, Kaho; Kubo, Shizuka; Miyazawa, Masami; Shirato, Saya; Hiroi, Yuka; Shiozawa, Kota

    2016-07-29

    It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Chromium downregulates the expression of Acetyl CoA Carboxylase 1 gene in lipogenic tissues of domestic goats: a potential strategy for meat quality improvement.

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    Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Zali, Abolfazl; Moradi-Shahrebabak, Hossein; Mousapour, Hojatollah

    2014-06-15

    Acetyl CoA Carboxylase 1 (ACC1) is a biotin-dependent enzyme that catalyzes the carboxylation of Acetyl CoA to form Malonyl CoA, the key intermediate metabolite in fatty acid synthesis. In this study, the mRNA expression of the ACC1 gene was evaluated in four different tissues (liver, visceral fat, subcutaneous fat, and longissimus muscle) of the domestic goat (Capra hircus) kids feeding on four different levels of trivalent chromium (0, 0.5, 1, and 1.5mg/day) as food supplementation. RT-qPCR technique was used for expression analyses and heat shock protein 90 gene (HSP-90) was considered as reference gene for data normalization. Our results revealed that 1.5mg/day chromium significantly reduced the expression of the ACC1 gene in liver, visceral fat, and subcutaneous fat tissues, but not in longissimus muscles (Pmeat quality in domestic animals. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Acetyl CoA Carboxylase Inhibition Reduces Hepatic Steatosis but Elevates Plasma Triglycerides in Mice and Humans: A Bedside to Bench Investigation.

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    Kim, Chai-Wan; Addy, Carol; Kusunoki, Jun; Anderson, Norma N; Deja, Stanislaw; Fu, Xiaorong; Burgess, Shawn C; Li, Cai; Ruddy, Marcie; Chakravarthy, Manu; Previs, Steve; Milstein, Stuart; Fitzgerald, Kevin; Kelley, David E; Horton, Jay D

    2017-08-01

    Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Prebiotic Fiber Increases Hepatic Acetyl CoA Carboxylase Phosphorylation and Suppresses Glucose-Dependent Insulinotropic Polypeptide Secretion More Effectively When Used with Metformin in Obese Rats1,2

    Science.gov (United States)

    Pyra, Kim A.; Saha, Dolan C.; Reimer, Raylene A.

    2013-01-01

    Independently, metformin (MET) and the prebiotic, oligofructose (OFS), have been shown to increase glucagon-like peptide (GLP-1) secretion. Our objective was to determine whether using OFS as an adjunct with MET augments GLP-1 secretion in obese rats. Male, diet-induced obese Sprague Dawley rats were randomized to: 1) high-fat/-sucrose diet [HFHS; control (C); 20% fat, 50% sucrose wt:wt]; 2) HFHS+10% OFS (OFS); 3) HFHS + MET [300 mg/kg/d (MET)]; 4) HFHS+10% OFS+MET (OFS +MET). Body composition, glycemia, satiety hormones, and mechanisms related to dipeptidyl peptidase 4 (DPP4) activity in plasma, hepatic AMP-activated protein kinase (AMPK; Western blots), and gut microbiota (qPCR) were examined. Direct effects of MET and SCFA were examined in human enteroendocrine cells. The interaction between OFS and MET affected fat mass, hepatic TG, secretion of glucose-dependent insulinotropic polypeptide (GIP) and leptin, and AMPKα2 mRNA and phosphorylated acetyl CoA carboxylase (pACC) levels (P < 0.05). Combined, OFS and MET reduced GIP secretion to a greater extent than either treatment alone (P < 0.05). The hepatic pACC level was increased by OFS+MET by at least 50% above all other treatments, which did not differ from each other (P < 0.05). OFS decreased plasma DPP4 activity (P < 0.001). Cecal Bifidobacteria (P < 0.001) were markedly increased and C. leptum decreased (P < 0.001) with OFS consumption. In human enteroendocrine cells, the interaction between MET and SCFA affected GLP-1 secretion (P < 0.04) but was not associated with higher GLP-1 than the highest individual doses. In conclusion, the combined actions of OFS and MET were associated with important interaction effects that have the potential to improve metabolic outcomes associated with obesity. PMID:22223580

  8. Effect of food deprivation and hormones of glucose homeostasis on the acetyl CoA carboxylase activity in mouse brain: a potential role of acc in the regulation of energy balance

    Directory of Open Access Journals (Sweden)

    Mukherjee Amrita

    2006-02-01

    Full Text Available Abstract We studied the regulation of brain acetyl CoA carboxylase (ACC activity during food deprivation and under the influence of hormones of glucose homeostasis: glucagon and insulin. Mice were deprived of food and water for time periods of 1, 3, 6, 9, 12 and 24 hours and were then allowed to re-feed for 5, 30 and 60 minutes. Mice that were deprived for up to 6 h, and then re-fed for 60 min, consumed the same amount of food compared to the ad libitum (control animals. However, after 9 h of deprivation, mice consumed only 50% of food present even after 1 h of re-feeding, compared to the controls. The ACC activity was measured in the whole mouse brain of controls and after 1, 3, 6, 9, 12, and 24 h of food deprivation. Brain extracts assayed from control mice expressed an ACC activity of 0.988 ± 0.158 fmol/min/mg tissue without citrate and 0.941 ± 0.175 fmol/min/mg tissue with citrate. After 1 h of food deprivation, the total ACC activity without citrate decreased to 0.575 ± 0.087 fmol/min/mg and in the presence of citrate, 0.703 ± 0.036 fmol/min/mg activity was measured. The citrate-dependent ACC activity decreased over time, with only 0.478 ± 0.117 fmol/min/mg of activity remaining after 24 h. Intraperitoneal (i.p. injections of insulin, glucagon and phosphate buffered saline (PBS were performed and whole brain ACC activity measured. After hormone administration, there were no significant differences in ACC activity in the presence of citrate. However, in the absence of citrate, there was a significant 20% decrease in ACC activity with glucagon (1.36 ± 0.09 fmol/min/mg and a 33% increase with insulin (2.49 ± 0.11 fmol/min/mg injections compared to PBS controls (1.67 ± 0.08 fmol/min/mg. Neuropeptide Y (NPY levels of corresponding brain extracts were measured by ELISA (OD using anti-NPY antibody and showed an 18% decrease upon insulin injection (0.093 ± 0.019 and a 50% increase upon glucagon injection (0.226 ± 0.084 as compared to

  9. The dynamic organization of fungal acetyl-CoA carboxylase

    Science.gov (United States)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  10. Induction of Cytosolic Acetyl-Coenzyme A Carboxylase in Pea Leaves by Ultraviolet-B Irradiation

    OpenAIRE

    Tomokazu, Konishi; Takahiro, Kamoi; Ryuichi, Matsuno; Yukiko, Sasaki; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Molecular Genetics, Biotechnology Institute, Akita Prefectural College of Agriculture; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Plant Molecular Biology, School of Agricultural Sciences, Nagoya University

    1996-01-01

    Levels of subunits of two acetyl-coenzyme A carboxylases were high in small leaves of Pisum sativum, decreased with growth, and remained constant in fully expanded leaves. Irradiation of fully expanded leaves induced the cytosolic isozyme only. This result suggests a key role for the cytosolic enzyme in protection against UV-B.

  11. Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase.

    Science.gov (United States)

    Westerhold, Lauren E; Bridges, Lance C; Shaikh, Saame Raza; Zeczycki, Tonya N

    2017-07-11

    Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

  12. A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation.

    Science.gov (United States)

    Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan; Sadre-Bazzaz, Kianoush; Rudolph, Michael J; Amodeo, Gabriele A; Symington, Lorraine S; Walz, Thomas; Tong, Liang

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3-AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery.

  13. Insect acetyl-CoA carboxylase: activity during the larval, pupal and adult stages of insect development.

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    Goldring, J P; Read, J S

    1993-12-01

    1. The activity of the lipogenic enzyme, acetyl-CoA carboxylase, was investigated in four insect species; Bombyx mori (Lepidoptera), Tenebrio molitor (Coleoptera), Glossina morsitans and Sarcophaga nodosa (Diptera). 2. Acetyl-CoA carboxylase activity in larval, pupal and adult forms was compared with the saponifiable lipid mass at each stage of the life-cycle, and found to follow similar patterns except for Tenebrio molitor. 3. The results are examined in relation to known metabolic requirements for each insect.

  14. Molecular Cloning and Characterization of Two Genes for the Biotin Carboxylase and Carboxyltransferase Subunits of Acetyl Coenzyme A Carboxylase in Myxococcus xanthus

    OpenAIRE

    Kimura, Yoshio; Miyake, Rina; Tokumasu, Yushi; Sato, Masayuki

    2000-01-01

    We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar t...

  15. Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Jia; Tong, Liang

    2015-10-12

    Acetyl-CoA carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases1, 2, 3, 4, 5, 6. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope7, 8. ACC contains biotin carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250-kilodalton (kDa), multi-domain enzymes and function as homodimers and higher oligomers. They contain a unique, 80-kDa central region that shares no homology with other proteins. Although the structures of the BC, CT and BCCP domains and other biotin-dependent carboxylase holoenzymes are known1, 9, 10, 11, 12, 13, 14, there is currently no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500-kDa holoenzyme dimer of ScACC. The structure is remarkably different from that of the other biotin-dependent carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of the BC domain alone, which is a monomer. These structural changes reveal why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A15, 16 and by phosphorylation of a Ser residue just before the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 Å, and the entire BCCP domain must translocate during catalysis.

  16. Evidence against translational repression by the carboxyltransferase component of Escherichia coli acetyl coenzyme A carboxylase.

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    Smith, Alexander C; Cronan, John E

    2014-11-01

    In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217-1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Acetyl-CoA carboxylase-a as a novel target for cancer therapy.

    Science.gov (United States)

    Wang, Chun; Rajput, Sandeep; Watabe, Kounosuke; Liao, Duan-Fang; Cao, Deliang

    2010-01-01

    Acetyl-CoA carboxylases (ACC) are rate-limiting enzymes in de novo fatty acid synthesis, catalyzing ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA. Malonyl-CoA is a critical bi-functional molecule, i.e., a substrate of fatty acid synthase (FAS) for acyl chain elongation (fatty acid synthesis) and an inhibitor of carnitine palmitoyltransferase I (CPT-I) for fatty acid beta-oxidation. Two ACC isoforms have been identified in mammals, i.e. ACC-alpha (ACCA, also termed ACC1) and ACC-beta (ACCB, also designated ACC2). ACC has long been used as a target for the management of metabolic diseases, such as obesity and metabolic syndrome, and various inhibitors have been developed in clinical trials. Recently, ACCA up-regulation has been recognized in multiple human cancers, promoting lipogenesis to meet the need of cancer cells for rapid growth and proliferation. Therefore, ACCA might be effective as a potent target for cancer intervention, and the inhibitors developed for the treatment of metabolic diseases would be potential therapeutic agents for cancer therapy. This review summarizes our recent findings and updates the current understanding of the ACCA with focus on cancer research.

  18. Cloning and expression analysis of carboxyltransferase of acetyl-coA carboxylase from Jatropha curcas.

    Science.gov (United States)

    Xie, Wu-Wei; Gao, Shun; Wang, Sheng-Hua; Zhu, Jin-Qiu; Xu, Ying; Tang, Lin; Chen, Fang

    2010-01-01

    A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl-CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fluorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased significantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a significant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fluctuations compared to those of the control. These findings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

  19. Synthesis of O-[11C]acetyl CoA, O-[11C]acetyl-L-carnitine, and L-[11C]carnitine labelled in specific positions, applied in PET studies on rhesus monkey

    International Nuclear Information System (INIS)

    Jacobson, Gunilla B.; Watanabe, Yasuyoshi; Valind, Sven; Kuratsune, Hirohiko; Laangstroem, Bengt

    1997-01-01

    The syntheses of L-carnitine, O-acetyl CoA, and O-acetyl-L-carnitine labelled with 11 C at the 1- or 2-position of the acetyl group or the N-methyl position of carnitine, using the enzymes acetyl CoA synthetase and carnitine acetyltransferase, are described. With a total synthesis time of 45 min, O-[1- 11 C]acetyl CoA and O-[2- 11 C]acetyl CoA was obtained in 60-70% decay-corrected radiochemical yield, and O-[1- 11 C]acetyl-L-carnitine and O-[2- 11 C]acetyl-L-carnitine in 70-80% yield, based on [1- 11 C]acetate or [2- 11 C]acetate, respectively. By an N-methylation reaction with [ 11 C]methyl iodide, L-[methyl- 11 C]carnitine was obtained within 30 min, and O-acetyl-L-[methyl- 11 C]carnitine within 40 min, giving a decay-corrected radiochemical yield of 60% and 40-50%, respectively, based on [ 11 C]methyl iodide. Initial data of the kinetics of the different 11 C-labelled L-carnitine and acetyl-L-carnitines in renal cortex of anaesthetized monkey (Macaca mulatta) are presented

  20. Synthesis of O-[{sup 11}C]acetyl CoA, O-[{sup 11}C]acetyl-L-carnitine, and L-[{sup 11}C]carnitine labelled in specific positions, applied in PET studies on rhesus monkey

    Energy Technology Data Exchange (ETDEWEB)

    Jacobson, Gunilla B.; Watanabe, Yasuyoshi; Valind, Sven; Kuratsune, Hirohiko; Laangstroem, Bengt

    1997-07-01

    The syntheses of L-carnitine, O-acetyl CoA, and O-acetyl-L-carnitine labelled with {sup 11}C at the 1- or 2-position of the acetyl group or the N-methyl position of carnitine, using the enzymes acetyl CoA synthetase and carnitine acetyltransferase, are described. With a total synthesis time of 45 min, O-[1-{sup 11}C]acetyl CoA and O-[2-{sup 11}C]acetyl CoA was obtained in 60-70% decay-corrected radiochemical yield, and O-[1-{sup 11}C]acetyl-L-carnitine and O-[2-{sup 11}C]acetyl-L-carnitine in 70-80% yield, based on [1-{sup 11}C]acetate or [2-{sup 11}C]acetate, respectively. By an N-methylation reaction with [{sup 11}C]methyl iodide, L-[methyl-{sup 11}C]carnitine was obtained within 30 min, and O-acetyl-L-[methyl-{sup 11}C]carnitine within 40 min, giving a decay-corrected radiochemical yield of 60% and 40-50%, respectively, based on [{sup 11}C]methyl iodide. Initial data of the kinetics of the different {sup 11}C-labelled L-carnitine and acetyl-L-carnitines in renal cortex of anaesthetized monkey (Macaca mulatta) are presented.

  1. AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

    Science.gov (United States)

    Soumya, Neelagiri; Kumar, I Sravan; Shivaprasad, S; Gorakh, Landage Nitin; Dinesh, Neeradi; Swamy, Kayala Kambagiri; Singh, Sushma

    2015-04-01

    An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Cell cycle regulation of the BRCA1/acetyl-CoA-carboxylase complex.

    Science.gov (United States)

    Ray, H; Suau, F; Vincent, A; Dalla Venezia, N

    2009-01-16

    Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. The BRCA1 protein has been implicated in multiple cellular functions. We have recently demonstrated that BRCA1 reduces acetyl-CoA-carboxylase alpha (ACCA) activity through its phospho-dependent binding to ACCA, and further established that the phosphorylation of the Ser1263 of ACCA is required for this interaction. Here, to gain more insight into the cellular conditions that trigger the BRCA1/ACCA interaction, we designed an anti-pSer1263 antibody and demonstrated that the Ser1263 of ACCA is phosphorylated in vivo, in a cell cycle-dependent manner. We further showed that the interaction between BRCA1 and ACCA is regulated during cell cycle progression. Taken together, our findings reveal a novel mechanism of regulation of ACCA distinct from the previously described phosphorylation of Ser79, and provide new insights into the control of lipogenesis through the cell cycle.

  3. BRCA1 affects lipid synthesis through its interaction with acetyl-CoA carboxylase.

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    Moreau, Karen; Dizin, Eva; Ray, Hind; Luquain, Céline; Lefai, Etienne; Foufelle, Fabienne; Billaud, Marc; Lenoir, Gilbert M; Venezia, Nicole Dalla

    2006-02-10

    Germ line alterations in BRCA1 (breast cancer susceptibility gene 1) are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 acts as a scaffold protein implicated in multiple cellular functions, such as transcription, DNA repair, and ubiquitination. However, the molecular mechanisms responsible for tumorigenesis are not yet fully understood. We have recently demonstrated that BRCA1 interacts in vivo with acetyl coenzyme A carboxylase alpha (ACCA) through its tandem of BRCA1 C terminus (BRCT) domains. To understand the biological function of the BRCA1.ACCA complex, we sought to determine whether BRCA1 is a regulator of lipogenesis through its interaction with ACCA. We showed here that RNA inhibition-mediated down-regulation of BRCA1 expression induced a marked increase in the fatty acid synthesis. We then delineated the biochemical characteristics of the complex and found that BRCA1 interacts solely with the phosphorylated and inactive form of ACCA (P-ACCA). Finally, we demonstrated that BRCA1 affects lipid synthesis by preventing P-ACCA dephosphorylation. These results suggest that BRCA1 affects lipogenesis through binding to P-ACCA, providing a new mechanism by which BRCA1 may exert a tumor suppressor function.

  4. Acetyl-CoA Carboxylase-α Inhibitor TOFA Induces Human Cancer Cell Apoptosis

    Science.gov (United States)

    Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-fang; Cao, Deliang

    2009-01-01

    Acetyl-CoA carboxylase-α (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC50 at approximately 5.0, 5.0, and 4.5 μg/ml, respectively. TOFA at 1.0–20.0 μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis. PMID:19450551

  5. Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.

    Science.gov (United States)

    Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang

    2009-07-31

    Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

  6. BRCA1 and acetyl-CoA carboxylase: the metabolic syndrome of breast cancer.

    Science.gov (United States)

    Brunet, Joan; Vazquez-Martin, Alejandro; Colomer, Ramon; Graña-Suarez, Begoña; Martin-Castillo, Begoña; Menendez, Javier A

    2008-02-01

    Breast cancer-associated mutations affecting the highly-conserved C-terminal BRCT domains of the tumor suppressor gene breast cancer susceptibility gene 1 (BRCA1) fully disrupt the ability of BRCA1 to interact with acetyl coenzyme A carboxylase alpha (ACCA), the rate-limiting enzyme catalyzing de novo fatty acid biogenesis. Specifically, BRCA1 interacts solely with the phosphorylated (inactive) form of ACCA (P-ACCA), and the formation of the BRCA1/P-ACCA complex interferes with ACCA activity by preventing P-ACCA dephosphorylation. One of the hallmarks of aggressive cancer cells is a high rate of energy-consuming anabolic processes driving the synthesis of lipids, proteins, and DNA (all of which are regulated by the energy status of the cell). The ability of BRCA1 to stabilize the phosphorylated/inactive form of ACCA strongly suggests that the tumor suppressive function of BRCA1 closely depends on its ability to mimic a cellular-low-energy status, which is known to block tumor cell anabolism and suppress the malignant phenotype. Interestingly, physical exercise and lack of obesity in adolescence have been associated with significantly delayed breast cancer onset for Ashkenazi Jewish women carrying BRCA1 gene mutations. Further clinical work may explore a chemopreventative role of "low-energy-mimickers" deactivating the ACCA-driven "lipogenic phenotype" in women with inherited mutations in BRCA1. This goal might be obtained with current therapeutic approaches useful in treating the metabolic syndrome and associated disorders in humans (e.g., type 2 diabetes and obesity), including metformin, thiazolidinediones (TZDs), calorie deprivation, and exercise. Alternatively, new forthcoming ACCA inhibitors may be relevant in the management of BRCA1-dependent breast cancer susceptibility and development. (c) 2007 Wiley-Liss, Inc.

  7. Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA▿

    OpenAIRE

    Uchino, Keiichi; Saito, Terumi; Gebauer, Birgit; Jendrossek, Dieter

    2007-01-01

    Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in...

  8. Determination of ploidy level and isolation of genes encoding acetyl-CoA carboxylase in Japanese Foxtail (Alopecurus japonicus.

    Directory of Open Access Journals (Sweden)

    Hongle Xu

    Full Text Available Ploidy level is important in biodiversity studies and in developing strategies for isolating important plant genes. Many herbicide-resistant weed species are polyploids, but our understanding of these polyploid weeds is limited. Japanese foxtail, a noxious agricultural grass weed, has evolved herbicide resistance. However, most studies on this weed have ignored the fact that there are multiple copies of target genes. This may complicate the study of resistance mechanisms. Japanese foxtail was found to be a tetraploid by flow cytometer and chromosome counting, two commonly used methods in the determination of ploidy levels. We found that there are two copies of the gene encoding plastidic acetyl-CoA carboxylase (ACCase in Japanese foxtail and all the homologous genes are expressed. Additionally, no difference in ploidy levels or ACCase gene copy numbers was observed between an ACCase-inhibiting herbicide-resistant and a herbicide-sensitive population in this study.

  9. Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

    Science.gov (United States)

    Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

    2010-01-01

    Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

  10. Activation of acetyl-coenzyme A carboxylase is involved in Taxol-induced ovarian cancer cell death.

    Science.gov (United States)

    Wu, Jiang; Ji, Fang; DI, Wen; Chen, Hongduo; Wan, Yinsheng

    2011-05-01

    Acetyl-coenzyme A carboxylase (ACC) is an attractive target for research into the treatment of a variety of human diseases, including diabetes, obesity and cancer. Mounting evidence suggests that the inhibition of ACC induced of cancer cell apoptosis. However, whether the inhibition of ACC regulates apoptosis in CaOV3 cancer cells has yet to be addressed. This study investigated the cytotoxic mechanism of action of ACC inhibition. Results showed that 5-(tetradecyloxy)-2-furoic acid (TOFA), an ACC inhibitor, enhanced Taxol-induced CaOV3 human ovarian cancer cell apoptosis. Notably, when TOFA was administered as a monotherapy, it induced CaOV3 cell apoptosis. Pre-treatment with the EGFR inhibitor PD153035 was found to markedly enhance ACC phosphorylation, whereas AMP-activated protein kinase (AMPK) activator AICAR was found to marginally enhance ACC phosphorylation. Taken together, the data showed ACC is a potential novel molecular target of Taxol. Additionally, ACC inhibition partially contributed to the cytotoxic effect of Taxol in ovarian cancer cells.

  11. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Science.gov (United States)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  12. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  13. Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase.

    Science.gov (United States)

    Chen, Jianguo; Jeppesen, Per Bendix; Nordentoft, Iver; Hermansen, Kjeld

    2007-06-01

    Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.

  14. Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

    Science.gov (United States)

    Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

    1992-01-01

    Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

  15. Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant.

    Science.gov (United States)

    Bandaranayake, Pradeepa C G; Yoder, John I

    2013-05-01

    Parasitic species of the family Orobanchaceae are devastating agricultural pests in many parts of the world. The control of weedy Orobanchaceae spp. is challenging, particularly due to the highly coordinated life cycles of the parasite and host plants. Although host genetic resistance often provides the foundation of plant pathogen management, few genes that confer resistance to root parasites have been identified and incorporated into crop species. Members of the family Orobanchaceae acquire water, nutrients, macromolecules, and oligonucleotides from host plants through haustoria that connect parasite and host plant roots. We are evaluating a resistance strategy based on using interfering RNA (RNAi) that is made in the host but inhibitory in the parasite as a parasite-derived oligonucleotide toxin. Sequences from the cytosolic acetyl-CoA carboxylase (ACCase) gene from Triphysaria versicolor were cloned in hairpin conformation and introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation. Transgenic roots were recovered for four of five ACCase constructions and infected with T. versicolor against parasitic weeds. In all cases, Triphysaria root viability was reduced up to 80% when parasitizing a host root bearing the hairpin ACCase. Triphysaria root growth was recovered by exogenous application of malonate. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that ACCase transcript levels were dramatically decreased in Triphysaria spp. parasitizing transgenic Medicago roots. Northern blot analysis identified a 21-nucleotide, ACCase-specific RNA in transgenic M. truncatula and in T. versicolor attached to them. One hairpin ACCase construction was lethal to Medicago spp. unless grown in media supplemented with malonate. Quantitative RT-PCR showed that the Medicago ACCase was inhibited by the Triphysaria ACCase RNAi. This work shows that ACCase is an effective target for inactivation in parasitic plants by trans-specific gene

  16. Abundance and distribution of archaeal acetyl-CoA/propionyl-CoA carboxylase genes indicative for putatively chemoautotrophic Archaea in the tropical Atlantic's interior

    OpenAIRE

    Bergauer, Kristin; Sintes, Eva; van Bleijswijk, Judith; Witte, Harry; Herndl, Gerhard J; Lueders, Tillmann

    2013-01-01

    Recently, evidence suggests that dark CO2 fixation in the pelagic realm of the ocean does not only occur in the suboxic and anoxic water bodies but also in the oxygenated meso- and bathypelagic waters of the North Atlantic. To elucidate the significance and phylogeny of the key organisms mediating dark CO2 fixation in the tropical Atlantic, we quantified functional genes indicative for CO2 fixation. We used a Q-PCR-based assay targeting the bifunctional acetyl-CoA/propionyl-CoA carboxylase (a...

  17. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    Energy Technology Data Exchange (ETDEWEB)

    Adam, Tasneem; Opie, Lionel H. [Hatter Cardiovascular Research Institute, Faculty of Health Sciences, University of Cape Town, Observatory 7925 (South Africa); Essop, M. Faadiel, E-mail: mfessop@sun.ac.za [Cardio-Metabolic Research Group (CMRG), Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600 (South Africa)

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  18. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Witters, L.A.; Bacon, G.W.

    1985-01-01

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

  19. FGF21 does not require adipocyte AMP-activated protein kinase (AMPK) or the phosphorylation of acetyl-CoA carboxylase (ACC) to mediate improvements in whole-body glucose homeostasis

    DEFF Research Database (Denmark)

    Mottillo, Emilio P; Desjardins, Eric M; Fritzen, Andreas Mæchel

    2017-01-01

    1β2AKO) and littermate controls were fed a high fat diet (HFD) and treated with native FGF21 or saline for two weeks. Additionally, HFD-fed mice with knock-in mutations on the AMPK phosphorylation sites of acetyl-CoA carboxylase (ACC)1 and ACC2 (DKI mice) along with wild-type (WT) controls received...

  20. Accumulation fatty acids of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense

    Science.gov (United States)

    Leyva, Luis A.; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E.

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  1. Genome-Wide Identification and Expression Analysis of the Biotin Carboxyl Carrier Subunits of Heteromeric Acetyl-CoA Carboxylase in Gossypium

    Directory of Open Access Journals (Sweden)

    Jinping Hua

    2017-05-01

    Full Text Available Acetyl-CoA carboxylase is an important enzyme, which catalyzes acetyl-CoA’s carboxylation to produce malonyl-CoA and to serve as a committed step for de novo fatty acid biosynthesis in plastids. In this study, 24 putative cotton BCCP genes were identified based on the lately published genome data in Gossypium. Among them, 4, 4, 8, and 8 BCCP homologs were identified in Gossypium raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. These genes were divided into two classes based on a phylogenetic analysis. In each class, these homologs were relatively conserved in gene structure and motifs. The chromosomal distribution pattern revealed that all the BCCP genes were distributed equally on corresponding chromosomes or scaffold in the four cotton species. Segmental duplication was a predominant duplication event in both of G. hirsutum and G. barbadense. The analysis of the expression profile showed that 8 GhBCCP genes expressed in all the tested tissues with changed expression levels, and GhBCCP genes belonging to class II were predominantly expressed in developing ovules. Meanwhile, the expression analysis for the 16 cotton BCCP genes from G. raimondii, G. arboreum and G. hirsutum showed that they were induced or suppressed by cold or salt stress, and their expression patterns varied among different tissues. These findings will help to determine the functional and evolutionary characteristics of the BCCP genes in Gossypium species.

  2. Modification of the Host Cell Lipid Metabolism Induced by Hypolipidemic Drugs Targeting the Acetyl Coenzyme A Carboxylase Impairs West Nile Virus Replication.

    Science.gov (United States)

    Merino-Ramos, Teresa; Vázquez-Calvo, Ángela; Casas, Josefina; Sobrino, Francisco; Saiz, Juan-Carlos; Martín-Acebes, Miguel A

    2016-01-01

    West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bite of mosquitoes that causes meningitis and encephalitis in humans, horses, and birds. Several studies have highlighted that flavivirus infection is highly dependent on cellular lipids for virus replication and infectious particle biogenesis. The first steps of lipid synthesis involve the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA that is catalyzed by the acetyl-CoA carboxylase (ACC). This makes ACC a key enzyme of lipid synthesis that is currently being evaluated as a therapeutic target for different disorders, including cancers, obesity, diabetes, and viral infections. We have analyzed the effect of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) on infection by WNV. Lipidomic analysis of TOFA-treated cells confirmed that this drug reduced the cellular content of multiple lipids, including those directly implicated in the flavivirus life cycle (glycerophospholipids, sphingolipids, and cholesterol). Treatment with TOFA significantly inhibited the multiplication of WNV in a dose-dependent manner. Further analysis of the antiviral effect of this drug showed that the inhibitory effect was related to a reduction of viral replication. Furthermore, treatment with another ACC inhibitor, 3,3,14,14-tetramethylhexadecanedioic acid (MEDICA 16), also inhibited WNV infection. Interestingly, TOFA and MEDICA 16 also reduced the multiplication of Usutu virus (USUV), a WNV-related flavivirus. These results point to the ACC as a druggable cellular target suitable for antiviral development against WNV and other flaviviruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Study on the Efficacy of Some Current Herbicides for Control of Resistant and Susceptible Canarygrass (Phalaris spp. Biotypes to Acetyl CoA Carboxylase (ACCase Inhibitors

    Directory of Open Access Journals (Sweden)

    e Zand

    2011-02-01

    Full Text Available Abstract Two separate greenhouse experiments were conducted in the greenhouse facilities of the Iranian Plant Protection Research Institute, Tehran, to study the efficacy of some herbicides to control of resistant and susceptible P. minor and P. paradoxa biotypes. In each experiment, resistant and susceptible biotypes were treated separately by 19 herbicide treatments. Treatments included 10 ACCase inhibitors, 6 Acetolactate Synthase (ALS inhibitors, prosulfocarb, flamprop-M-isopropyl, isoproturon plus diflufenican and a non-sprayed control. To evaluate the effects of treatments, different characteristics including percent damage based on EWRC scores at 15 and 30 days after spraying, percentage of survived plants after spraying relative to before spraying, and percentage of dry weight and wet weight of individual plants relative to control were studied. Results showed that the susceptible biotypes of P. minor were best controlled by clodinafop propargyl and pinoxaden at 450 ml/ha while pinoxaden at 450 ml/ha and cycloxydim were best options for control of the resistant biotype. Among ALS inhibitors, iodosulfuron plus mesosulfuron could control susceptible and resistant biotypes of P. minor very effectively and semi-satisfactory, respectively. Iodosulfuron plus mesosulfuron and sulfosulfuron plus metsulfuron could remarkably reduce the wet weight of individual plants compared to control so that the plants were not damaging any more. Among other herbicides, isoproturon plus diflufenican could control the susceptible and resistant biotypes semi-satisfactory and very effectively, respectively. Keywords: Herbicide resistance, ACCase inhibitors, ALS inhibitors

  4. Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Griffith, David A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Kung, Daniel W. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Esler, William P. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Amor, Paul A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Bagley, Scott W. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Beysen, Carine [KineMed Inc., Emeryville, CA (United States); Carvajal-Gonzalez, Santos [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Doran, Shawn D. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Limberakis, Chris [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Mathiowetz, Alan M. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); McPherson, Kirk [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Price, David A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Ravussin, Eric [Louisiana State Univ., Baton Rouge, LA (United States); Sonnenberg, Gabriele E. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Southers, James A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Sweet, Laurel J. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Turner, Scott M. [KineMed Inc., Emeryville, CA (United States); Vajdos, Felix F. [Pfizer Worldwide Research and Development, Cambridge, MA (United States)

    2014-12-26

    We found that Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. Here, we disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.

  5. Nuclear-cytoplasmic conflict in pea (Pisum sativum L. is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

    Directory of Open Access Journals (Sweden)

    Vera S Bogdanova

    Full Text Available In crosses of wild and cultivated peas (Pisum sativum L., nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

  6. Nuclear-Cytoplasmic Conflict in Pea (Pisum sativum L.) Is Associated with Nuclear and Plastidic Candidate Genes Encoding Acetyl-CoA Carboxylase Subunits

    Science.gov (United States)

    Bogdanova, Vera S.; Zaytseva, Olga O.; Mglinets, Anatoliy V.; Shatskaya, Natalia V.; Kosterin, Oleg E.; Vasiliev, Gennadiy V.

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized. PMID:25789472

  7. Screening Phosphorylation Site Mutations in Yeast Acetyl-CoA Carboxylase Using Malonyl-CoA Sensor to Improve Malonyl-CoA-Derived Product.

    Science.gov (United States)

    Chen, Xiaoxu; Yang, Xiaoyu; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2018-01-01

    Malonyl-coenzyme A (malonyl-CoA) is a critical precursor for the biosynthesis of a variety of biochemicals. It is synthesized by the catalysis of acetyl-CoA carboxylase (Acc1p), which was demonstrated to be deactivated by the phosphorylation of Snf1 protein kinase in yeast. In this study, we designed a synthetic malonyl-CoA biosensor and used it to screen phosphorylation site mutations of Acc1p in Saccharomyces cerevisiae . Thirteen phosphorylation sites were mutated, and a combination of three site mutations in Acc1p, S686A, S659A, and S1157A, was found to increase malonyl-CoA availability. ACC1 S686AS659AS1157A expression also improved the production of 3-hydroxypropionic acid, a malonyl-CoA-derived chemical, compared to both wild type and the previously reported ACC1 S659AS1157A mutation. This mutation will also be beneficial for other malonyl-CoA-derived products.

  8. Maternal obesity reduces milk lipid production in lactating mice by inhibiting acetyl-CoA carboxylase and impairing fatty acid synthesis.

    Science.gov (United States)

    Saben, Jessica L; Bales, Elise S; Jackman, Matthew R; Orlicky, David; MacLean, Paul S; McManaman, James L

    2014-01-01

    Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.

  9. Acetyl-CoA carboxylase rewires cancer metabolism to allow cancer cells to survive inhibition of the Warburg effect by cetuximab.

    Science.gov (United States)

    Luo, Jingtao; Hong, Yun; Lu, Yang; Qiu, Songbo; Chaganty, Bharat K R; Zhang, Lun; Wang, Xudong; Li, Qiang; Fan, Zhen

    2017-01-01

    Cetuximab inhibits HIF-1-regulated glycolysis in cancer cells, thereby reversing the Warburg effect and leading to inhibition of cancer cell metabolism. AMP-activated protein kinase (AMPK) is activated after cetuximab treatment, and a sustained AMPK activity is a mechanism contributing to cetuximab resistance. Here, we investigated how acetyl-CoA carboxylase (ACC), a downstream target of AMPK, rewires cancer metabolism in response to cetuximab treatment. We found that introduction of experimental ACC mutants lacking the AMPK phosphorylation sites (ACC1_S79A and ACC2_S212A) into head and neck squamous cell carcinoma (HNSCC) cells protected HNSCC cells from cetuximab-induced growth inhibition. HNSCC cells with acquired cetuximab resistance contained not only high levels of T172-phosphorylated AMPK and S79-phosphorylated ACC1 but also an increased level of total ACC. These findings were corroborated in tumor specimens of HNSCC patients treated with cetuximab. Cetuximab plus TOFA (an allosteric inhibitor of ACC) achieved remarkable growth inhibition of cetuximab-resistant HNSCC xenografts. Our data suggest a novel paradigm in which cetuximab-mediated activation of AMPK and subsequent phosphorylation and inhibition of ACC is followed by a compensatory increase in total ACC, which rewires cancer metabolism from glycolysis-dependent to lipogenesis-dependent. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. The glossyhead1 allele of acc1 reveals a principal role for multidomain acetyl-coenzyme a carboxylase in the biosynthesis of cuticular waxes by Arabidopsis

    KAUST Repository

    Lu, Shiyou

    2011-09-23

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C 20:0 or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling. © 2011 American Society of Plant Biologists. All Rights Reserved.

  11. Abundance and distribution of archaeal acetyl-CoA/propionyl-CoA carboxylase genes indicative for putatively chemoautotrophic Archaea in the tropical Atlantic's interior.

    Science.gov (United States)

    Bergauer, Kristin; Sintes, Eva; van Bleijswijk, Judith; Witte, Harry; Herndl, Gerhard J

    2013-06-01

    Recently, evidence suggests that dark CO2 fixation in the pelagic realm of the ocean does not only occur in the suboxic and anoxic water bodies but also in the oxygenated meso- and bathypelagic waters of the North Atlantic. To elucidate the significance and phylogeny of the key organisms mediating dark CO2 fixation in the tropical Atlantic, we quantified functional genes indicative for CO2 fixation. We used a Q-PCR-based assay targeting the bifunctional acetyl-CoA/propionyl-CoA carboxylase (accA subunit), a key enzyme powering inter alia the 3-hydroxypropionate/4-hydroxybutyrate cycle (HP/HB) and the archaeal ammonia monooxygenase (amoA). Quantification of accA-like genes revealed a consistent depth profile in the upper mesopelagial with increasing gene abundances from subsurface layers towards the oxygen minimum zone (OMZ), coinciding with an increase in archaeal amoA gene abundance. Gene abundance profiles of metabolic marker genes (accA, amoA) were correlated with thaumarchaeal 16S rRNA gene abundances as well as CO2 fixation rates to link the genetic potential to actual rate measurements. AccA gene abundances correlated with archaeal amoA gene abundance throughout the water column (r(2)  = 0.309, P < 0.0001). Overall, a substantial genetic predisposition of CO2 fixation was present in the dark realm of the tropical Atlantic in both Archaea and Bacteria. Hence, dark ocean CO2 fixation might be more widespread among prokaryotes inhabiting the oxygenated water column of the ocean's interior than hitherto assumed. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  12. Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism.

    Science.gov (United States)

    Ronnebaum, Sarah M; Joseph, Jamie W; Ilkayeva, Olga; Burgess, Shawn C; Lu, Danhong; Becker, Thomas C; Sherry, A Dean; Newgard, Christopher B

    2008-05-23

    Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.

  13. Chronic Suppression of Acetyl-CoA Carboxylase 1 in β-Cells Impairs Insulin Secretion via Inhibition of Glucose Rather Than Lipid Metabolism*

    Science.gov (United States)

    Ronnebaum, Sarah M.; Joseph, Jamie W.; Ilkayeva, Olga; Burgess, Shawn C.; Lu, Danhong; Becker, Thomas C.; Sherry, A. Dean; Newgard, Christopher B.

    2008-01-01

    Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60–80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to β-cells suppressed [14C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy. PMID:18381287

  14. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Shiro Maeda

    2010-02-01

    Full Text Available It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A carboxylase beta (ACACB as a candidate for a susceptibility to diabetic nephropathy; the landmark SNP was found in the intron 18 of ACACB (rs2268388: intron 18 +4139 C > T, p = 1.4x10(-6, odds ratio = 1.61, 95% confidence interval [CI]: 1.33-1.96. The association of this SNP with diabetic nephropathy was examined in 9 independent studies (4 from Japan including the original study, one Singaporean, one Korean, and two European with type 2 diabetes. One case-control study involving European patients with type 1 diabetes was included. The frequency of the T allele for SNP rs2268388 was consistently higher among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8, odds ratio = 1.61, 95% Cl: 1.35-1.91. Rs2268388 was also associated with type 2 diabetes-associated end-stage renal disease (ESRD in European Americans (p = 6 x 10(-4, odds ratio = 1.61, 95% Cl: 1.22-2.13. Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including rs2268388, had significant enhancer activity in cultured human renal proximal tubular epithelial cells. Fragments corresponding to the disease susceptibility allele (T had higher enhancer activity than those of the major allele. These results suggest that ACACB is a strong candidate for conferring susceptibility for proteinuria in patients with type 2 diabetes.

  15. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Maeda, Shiro; Kobayashi, Masa-aki; Araki, Shin-ichi

    2010-01-01

    It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A ca...

  16. Insight into the molecular mechanism of yeast acetyl-coenzyme A carboxylase mutants F510I, N485G, I69E, E477R, and K73R resistant to soraphen A

    Science.gov (United States)

    Gao, Jian; Liang, Li; Chen, Qingqing; Zhang, Ling; Huang, Tonghui

    2018-02-01

    Acetyl-coenzyme A carboxylases (ACCs) is the first committed enzyme of fatty acid synthesis pathway. The inhibition of ACC is thought to be beneficial not only for diseases related to metabolism, such as type-2 diabetes, but also for infectious disease like bacterial infection disease. Soraphen A, a potent allosteric inhibitor of BC domain of yeast ACC, exhibit lower binding affinities to several yeast ACC mutants and the corresponding drug resistance mechanisms are still unknown. We report here a theoretical study of binding of soraphen A to wild type and yeast ACC mutants (including F510I, N485G, I69E, E477R, and K73R) via molecular dynamic simulation and molecular mechanics/generalized Born surface area free energy calculations methods. The calculated binding free energies of soraphen A to yeast ACC mutants are weaker than to wild type, which is highly consistent with the experimental results. The mutant F510I weakens the binding affinity of soraphen A to yeast ACC mainly by decreasing the van der Waals contributions, while the weaker binding affinities of Soraphen A to other yeast ACC mutants including N485G, I69E, E477R, and K73R are largely attributed to the decreased net electrostatic (ΔE ele + ΔG GB) interactions. Our simulation results could provide important insights for the development of more potent ACC inhibitors.

  17. Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl2.

    Science.gov (United States)

    Damiano, Fabrizio; Testini, Mariangela; Tocci, Romina; Gnoni, Gabriele V; Siculella, Luisa

    2018-04-01

    Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl 2 , up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Seker, Tamay; Møller, Kasper; Nielsen, Jens

    2005-01-01

    The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl Co......A, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we...... used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase...

  19. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    International Nuclear Information System (INIS)

    Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T.

    2006-01-01

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy β and originally identified CoA synthase, CoASy α. The transcript specific for CoASy β was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy β. In contrast to CoASy α, which shows ubiquitous expression, CoASy β is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation

  20. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

    Science.gov (United States)

    Broussard, Tyler C.; Pakhomova, Svetlana; Neau, David B.; Bonnot, Ross; Waldrop, Grover L.

    2015-01-01

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1′-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1′-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin. PMID:26020841

  1. Differences among Adult COAs and Adult Non-COAs on Levels of Self-Esteem, Depression, and Anxiety.

    Science.gov (United States)

    Dodd, David T.; Roberts, Richard L.

    1994-01-01

    Examined self-esteem, depression, and anxiety among 60 adult children of alcoholics (COAs) and 143 adult non-COAs. Subjects completed Children of Alcoholics Screening Test, demographic questionnaire, Beck Depression Inventory, State-Trait Anxiety Inventory, and Coopersmith Self-Esteem Inventory. Found no significant differences between COAs and…

  2. Assay of ribulose bisphosphate carboxylase

    International Nuclear Information System (INIS)

    Pike, C.; Berry, J.

    1987-01-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, [ 14 C]-NaHCO 3 , and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30 0 . The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number

  3. CoaSim Guile Manual — Using the Guile-based CoaSim Simulator

    DEFF Research Database (Denmark)

    Mailund, T

    2006-01-01

    CoaSim is a tool for simulating the coalescent process with recombination and geneconversion, under either constant population size or exponential population growth. It effectively constructs the ancestral recombination graph for a given number of chromosomes and uses this to simulate samples...

  4. Getting Started with CoaSim — An Introduction to the Simulator CoaSim

    DEFF Research Database (Denmark)

    Mailund, T

    2005-01-01

    CoaSim is a tool for simulating the coalescent process with recombination and geneconversion, under either constant population size or exponential population growth. It effectively constructs the ancestral recombination graph for a given number of chromosomes and uses this to simulate samples...

  5. Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

    Science.gov (United States)

    Brylinski, Michal; Waldrop, Grover L

    2014-04-02

    As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×10⁸ amino-oxazole derivatives. A subset of 9×10⁶ of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug

  6. Studies of vitamin K-dependent carboxylase

    International Nuclear Information System (INIS)

    Wood, G.M.

    1986-01-01

    Carboxylase was studied in detergent solubilized rat liver microsomes, using the peptide substrate Phe-Leu-[γ- 3 H]-Glu-Glu-Leu. Cleavage of the γ-C-H bond in Glu was measured as the release of 3 H from this peptide to water, carboxylation was measured as the incorporation of H 14 CO 3 -into the peptide, and KO formation was measured by an HPLC assay. All three products could be measured simultaneously, and this system was used to examine the effects of cyanide, manganese, tetrachloropyridinol, and Boc-SerP-SerP-Leu-OMe on the separate steps of the carboxylase reaction. Vitamin K-epoxide formation was studied separately from the other reactions, and it was found that in the absence of a Glu-containing substrate, carboxylase catalyzed the uncoupled formation of KO from KH 2 and O 2 . The stoichiometry of product formation (GLa, KO, and γ-protons) was measured, and the results obtained were all in agreement with the values predicted from the proposed mechanism. When all of the substrates were saturating, the stoichiometry of γ-C-H bond cleavage, carboxylation, and KO formation was 1:1:1

  7. CoAs: The line of 3 d demarcation

    Science.gov (United States)

    Campbell, Daniel J.; Wang, Limin; Eckberg, Chris; Graf, Dave; Hodovanets, Halyna; Paglione, Johnpierre

    2018-05-01

    Transition metal-pnictide compounds have received attention for their tendency to combine magnetism and unconventional superconductivity. Binary CoAs lies on the border of paramagnetism and the more complex behavior seen in isostructural CrAs, MnP, FeAs, and FeP. Here we report the properties of CoAs single crystals grown with two distinct techniques along with density functional theory calculations of its electronic structure and magnetic ground state. While all indications are that CoAs is paramagnetic, both experiment and theory suggest proximity to a ferromagnetic instability. Quantum oscillations are seen in torque measurements up to 31.5 T and support the calculated paramagnetic Fermiology.

  8. Vitamin K-dependent carboxylases from non-hepatic tissues

    NARCIS (Netherlands)

    Vermeer, C.; Hendrix, H.; Daemen, M.

    1982-01-01

    The presence of vitamin K-dependent carboxylase was investigated in the microsomal fraction of 20 different types of bovine tissue. Except for muscle, veins, lymphocytes and bone membrane, carboxylase was found in all these preparations, albeit in varying amounts. No differences could be detected

  9. Effect of [L-Carnitine] on acetyl-L-carnitine production by heart mitochondria

    International Nuclear Information System (INIS)

    Bieber, L.L.; Lilly, K.; Lysiak, W.

    1986-01-01

    The authors recently reported a large efflux of acetyl-L-carnitine from rat heart mitochondria during state 3 respiration with pyruvate as substrate both in the presence and absence of malate. In this series of experiments, the effect of the concentration of L-carnitine on the efflux of acetyl-L-carnitine and on the production of 14 CO 2 from 2- 14 C-pyruvate was determined. Maximum acetylcarnitine production (approximately 25 n moles/min/mg protein) was obtained at 3-5 mM L-carnitine in the absence of added malate. 14 CO 2 production decreased as the concentration of L-carnitine increased; it plateaued at 3-5 mM L-carnitine. These data indicate carnitine can stimulate flux of pyruvate through pyruvate dehydrogenase and can reduce flux of acetyl CoA through the Krebs cycle by acting as an acceptor of the acetyl moieties of acetyl CoA generated by pyruvate dehydrogenase

  10. Overexpression of a foxtail millet Acetyl-CoA carboxylase gene in ...

    African Journals Online (AJOL)

    Administrator

    2011-05-16

    May 16, 2011 ... form, usually located in the cytoplasm, is a “eukaryotic- type” multifunctional enzyme. In the grass family ... 25°C, after which they were transferred to resting medium (D basal medium, 20 gl–1 Suc, 10 gl–1 Glc, 0.85 ... Total RNA of the transgenic and untransformed plants was extracted according to the Trizol ...

  11. Role of Feedback Regulation of Pantothenate Kinase (CoaA) in Control of Coenzyme A Levels in Escherichia coli

    Science.gov (United States)

    Rock, Charles O.; Park, Hee-Won; Jackowski, Suzanne

    2003-01-01

    Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4′-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels. PMID:12754240

  12. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    Science.gov (United States)

    Desai, P.; Ashokaan, N.; Masud, J.; Pariti, A.; Nath, M.

    2015-03-01

    This article provides a comprehensive guide on the synthesis and characterization of superparamagnetic CoAs nanoparticles and elongated nanostructures with high blocking temperature, (TB), via hot-injection precipitation and solvothermal methods. Cobalt arsenides constitute an important family of magnetically active solids that find a variety of applications ranging from magnetic semiconductors to biomedical imaging. While the higher temperature hot-injection precipitation technique (300 °C) yields pure CoAs nanostructures, the lower temperature solvothermal method (200 °C) yields a mixture of CoAs nanoparticles along with other Co-based impurity phases. The synthesis in all these cases involved usage of triphenylarsine ((C6H5)3As) as the As precursor which reacts with solid Co2(CO)8 by ligand displacement to yield a single source precursor. The surfactant, hexadecylamine (HDA) further assists in controlling the morphology of the nanostructures. HDA also provides a basic medium and molten flux-like conditions for the redox chemistry to occur between Co and As at elevated temperatures. The influence of the length of reaction time was investigated by studying the evolution of product morphology over time. It was observed that while spontaneous nucleation at higher temperature followed by controlled growth led to the predominant formation of short nanorods, with longer reaction time, the nanorods were further converted to nanoparticles. The size of the nanoparticles obtained, was mostly in the range of 10-15 nm. The key finding of this work is exceptionally high coercivity in CoAs nanostructures for the first time. Coercivity observed was as high as 0.1 T (1000 Oe) at 2 K. These kinds of magnetic nanostructures find multiple applications in spintronics, whereas the superparamagnetic nanoparticles are viable for use in magnetic storage, ferrofluids and as contrast enhancing agents in MRI.

  13. COA based robust output feedback UPFC controller design

    Energy Technology Data Exchange (ETDEWEB)

    Shayeghi, H., E-mail: hshayeghi@gmail.co [Technical Engineering Department, University of Mohaghegh Ardabili, Ardabil (Iran, Islamic Republic of); Shayanfar, H.A. [Center of Excellence for Power System Automation and Operation, Electrical Engineering Department, Iran University of Science and Technology, Tehran (Iran, Islamic Republic of); Jalilzadeh, S.; Safari, A. [Technical Engineering Department, Zanjan University, Zanjan (Iran, Islamic Republic of)

    2010-12-15

    In this paper, a novel method for the design of output feedback controller for unified power flow controller (UPFC) using chaotic optimization algorithm (COA) is developed. Chaotic optimization algorithms, which have the features of easy implementation, short execution time and robust mechanisms of escaping from the local optimum, is a promising tool for the engineering applications. The selection of the output feedback gains for the UPFC controllers is converted to an optimization problem with the time domain-based objective function which is solved by a COA based on Lozi map. Since chaotic mapping enjoys certainty, ergodicity and the stochastic property, the proposed chaotic optimization problem introduces chaos mapping using Lozi map chaotic sequences which increases its convergence rate and resulting precision. To ensure the robustness of the proposed stabilizers, the design process takes into account a wide range of operating conditions and system configurations. The effectiveness of the proposed controller for damping low frequency oscillations is tested and demonstrated through non-linear time-domain simulation and some performance indices studies. The results analysis reveals that the designed COA based output feedback UPFC damping controller has an excellent capability in damping power system low frequency oscillations and enhance greatly the dynamic stability of the power systems.

  14. Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

    Science.gov (United States)

    Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

    2011-01-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

  15. Pyruvate carboxylase is expressed in human skeletal muscle

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable...

  16. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, R.M.B. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia]|[Universidade Nova de Lisboa, Oeiras (Portugal). Instituto de Tecnologia Quimica e Biologica; Franco, E.; Teixeira, A.R.N. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of {sup 35}S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author).

  17. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    International Nuclear Information System (INIS)

    Ferreira, R.M.B.; Universidade Nova de Lisboa, Oeiras; Franco, E.; Teixeira, A.R.N.

    1996-01-01

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35 S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author)

  18. Properties of ribulose diphosphate carboxylase immobilized on porous glass

    Science.gov (United States)

    Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

    1974-01-01

    Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

  19. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  20. The human gastric pathogen Helicobacter pylori has a potential acetone carboxylase that enhances its ability to colonize mice

    Directory of Open Access Journals (Sweden)

    Weinberg Michael V

    2008-01-01

    Full Text Available Abstract Background Helicobacter pylori colonizes the human stomach and is the etiological agent of peptic ulcer disease. All three H. pylori strains that have been sequenced to date contain a potential operon whose products share homology with the subunits of acetone carboxylase (encoded by acxABC from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10. Acetone carboxylase catalyzes the conversion of acetone to acetoacetate. Genes upstream of the putative acxABC operon encode enzymes that convert acetoacetate to acetoacetyl-CoA, which is metabolized further to generate two molecules of acetyl-CoA. Results To determine if the H. pylori acxABC operon has a role in host colonization the acxB homolog in the mouse-adapted H. pylori SS1 strain was inactivated with a chloramphenicol-resistance (cat cassette. In mouse colonization studies the numbers of H. pylori recovered from mice inoculated with the acxB:cat mutant were generally one to two orders of magnitude lower than those recovered from mice inoculated with the parental strain. A statistical analysis of the data using a Wilcoxin Rank test indicated the differences in the numbers of H. pylori isolated from mice inoculated with the two strains were significant at the 99% confidence level. Levels of acetone associated with gastric tissue removed from uninfected mice were measured and found to range from 10–110 μmols per gram wet weight tissue. Conclusion The colonization defect of the acxB:cat mutant suggests a role for the acxABC operon in survival of the bacterium in the stomach. Products of the H. pylori acxABC operon may function primarily in acetone utilization or may catalyze a related reaction that is important for survival or growth in the host. H. pylori encounters significant levels of acetone in the stomach which it could use as a potential electron donor for microaerobic respiration.

  1. Role of an Essential Acyl Coenzyme A Carboxylase in the Primary and Secondary Metabolism of Streptomyces coelicolor A3(2)

    Science.gov (United States)

    Rodríguez, E.; Banchio, C.; Diacovich, L.; Bibb, M. J.; Gramajo, H.

    2001-01-01

    Two genes, accB and accE, that form part of the same operon, were cloned from Streptomyces coelicolor A3(2). AccB is homologous to the carboxyl transferase domain of several propionyl coezyme A (CoA) carboxylases and acyl-CoA carboxylases (ACCases) of actinomycete origin, while AccE shows no significant homology to any known protein. Expression of accB and accE in Escherichia coli and subsequent in vitro reconstitution of enzyme activity in the presence of the biotinylated protein AccA1 or AccA2 confirmed that AccB was the carboxyl transferase subunit of an ACCase. The additional presence of AccE considerably enhanced the activity of the enzyme complex, suggesting that this small polypeptide is a functional component of the ACCase. The impossibility of obtaining an accB null mutant and the thiostrepton growth dependency of a tipAp accB conditional mutant confirmed that AccB is essential for S. coelicolor viability. Normal growth phenotype in the absence of the inducer was restored in the conditional mutant by the addition of exogenous long-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. Thus, AccB, together with AccA2, which is also an essential protein (E. Rodriguez and H. Gramajo, Microbiology 143:3109–3119, 1999), are the most likely components of an ACCase whose main physiological role is the synthesis of malonyl-CoA, the first committed step of fatty acid synthesis. Although normal growth of the conditional mutant was restored by fatty acids, the cultures did not produce actinorhodin or undecylprodigiosin, suggesting a direct participation of this enzyme complex in the supply of malonyl-CoA for the synthesis of these secondary metabolites. PMID:11526020

  2. A Patient With Pyruvate Carboxylase Deficiency and Nemaline Rods on Muscle Biopsy

    DEFF Research Database (Denmark)

    Unal, Ozlem; Orhan, Diclehan; Ostergaard, Elsebet

    2013-01-01

    Nemaline rods are the pathologic hallmark of nemaline myopathy, but they have also been described as a secondary phenomenon in a variety of other disorders. Nemaline rods have not been reported in pyruvate carboxylase deficiency before. Here we present a patient with pyruvate carboxylase deficiency...

  3. The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel.

    NARCIS (Netherlands)

    Huberts, D.H.; Venselaar, H.; Vriend, G.; Veenhuis, M.; Klei, I.J. van der

    2010-01-01

    Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenulapolymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional

  4. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger; Zenger, Kyall R; Jerry, Dean R; Heimann, Kirsten

    2015-01-01

    as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases

  5. Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme.

    Science.gov (United States)

    Mukherjee, J J; Dekker, E E

    1987-10-25

    Starting with 100 g (wet weight) of a mutant of Escherichia coli K-12 forced to grow on L-threonine as sole carbon source, we developed a 6-step procedure that provides 30-40 mg of homogeneous 2-amino-3-ketobutyrate CoA ligase (also called aminoacetone synthetase or synthase). This ligase, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, has an apparent molecular weight approximately equal to 85,000 and consists of two identical (or nearly identical) subunits with Mr = 42,000. Computer analysis of amino acid composition data, which gives the best fit nearest integer ratio for each residue, indicates a total of 387 amino acids/subunit with a calculated Mr = 42,093. Stepwise Edman degradation provided the N-terminal sequence of the first 21 amino acids. It is a pyridoxal phosphate-dependent enzyme since (a) several carbonyl reagents caused greater than 90% loss of activity, (b) dialysis against buffer containing hydroxylamine resulted in 89% loss of activity coincident with an 86% decrease in absorptivity at 428 nm, (c) incubation of the apoenzyme with 20 microM pyridoxal phosphate showed a parallel recovery (greater than 90%) of activity and 428-nm absorptivity, and (d) reduction of the holoenzyme with NaBH4 resulted in complete inactivation, disappearance of a new absorption maximum at 333 nm. Strict specificity for glycine is shown but acetyl-CoA (100%), n-propionyl-CoA (127%), or n-butyryl-CoA (16%) is utilized in the condensation reaction. Apparent Km values for acetyl-CoA, n-propionyl-CoA, and glycine are 59 microM, 80 microM, and 12 mM, respectively; the pH optimum = 7.5. Added divalent metal ions or sulfhydryl compounds inhibited catalysis of the condensation reaction.

  6. Effect of elevated total CoA levels on metabolic pathways in cultured hepatocytes

    International Nuclear Information System (INIS)

    Steffen, C.A.; Smith, C.M.

    1987-01-01

    Livers from fasted rats have 30% higher total CoA levels than fed rats. To determine whether this increase of total CoA influences metabolism, the rates of gluconeogenesis, fatty acid oxidation and ketogenesis were measured in hepatocytes with cyanamide (CYM) or pantothenate (PA) deficient medium used to vary total CoA levels independently of hormonal status. Primary cultures of rat hepatocytes were incubated 14 hrs with Bt 2 cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 μM) + PA, rinsed and preincubated 0.5 hr to remove the CYM. Hepatocytes treated with CYM had total CoA levels 10-24% higher than PA deficient cells and lower rates of glucose production from lactate + pyruvate (L/P) or from alanine (0.23 +/- 0.05 and 0.089 +/- 0.02 μm/mg protein, respectively in CYM treated cells compared to 0.33 +/- 0.06 and 0.130 +/- 0.006 in PA deficient cells). This decrease was not due to CYM per se, as the direct addition of CYM stimulated glucose production from L/P. CYM treated cells with 15-40% higher total CoA and 30% higher fatty acyl-CoA levels had the same rates of [ 14 C]-palmitate oxidation as PA deficient cells. However, rates of ketogenesis were lower in CYM treated cells (163 +/- 11 nm/mg compared to 217 +/- 14 nm/mg protein). These results suggest that physiological alterations of hepatic total CoA levels are not necessary for fasting rates of gluconeogenesis, fatty acid oxidation and ketogenesis

  7. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    Science.gov (United States)

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. Anaplerotic roles of pyruvate carboxylase in mammalian tissues.

    Science.gov (United States)

    Jitrapakdee, S; Vidal-Puig, A; Wallace, J C

    2006-04-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC serves an anaplerotic role for the tricarboxylic acid cycle, when intermediates are removed for different biosynthetic purposes. In liver and kidney, PC provides oxaloacetate for gluconeogenesis. In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor-gamma, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis. In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the 'pyruvate/malate cycle' to shuttle 3C or 4C between mitochondria and cytoplasm. Hyperglycemia and hyperlipidemia impair this cycle and affect glucose-stimulated insulin release. In astrocytes, PC is important for de novo synthesis of glutamate, an important excitatory neurotransmitter supplied to neurons. Transcriptional studies of the PC gene pinpoint some transcription factors that determine tissue-specific expression.

  9. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    International Nuclear Information System (INIS)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-01-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. 14 C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell

  10. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    Energy Technology Data Exchange (ETDEWEB)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-05-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. /sup 14/C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell.

  11. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower propo...

  12. CoCoa: a software tool for estimating the coefficient of coancestry from multilocus genotype data.

    Science.gov (United States)

    Maenhout, Steven; De Baets, Bernard; Haesaert, Geert

    2009-10-15

    Phenotypic data collected in breeding programs and marker-trait association studies are often analyzed by means of linear mixed models. In these models, the covariance between the genetic background effects of all genotypes under study is modeled by means of pairwise coefficients of coancestry. Several marker-based coancestry estimation procedures allow to estimate this covariance matrix, but generally introduce a certain amount of bias when the examined genotypes are part of a breeding program. CoCoa implements the most commonly used marker-based coancestry estimation procedures and as such, allows to select the best fitting covariance structure for the phenotypic data at hand. This better model fit translates into an increased power and improved type I error control in association studies and an improved accuracy in phenotypic prediction studies. The presented software package also provides an implementation of the new Weighted Alikeness in State (WAIS) estimator for use in hybrid breeding programs. Besides several matrix manipulation tools, CoCoa implements two different bending heuristics, in case the inverse of an ill-conditioned coancestry matrix estimate is needed. The software package CoCoa is freely available at http://webs.hogent.be/cocoa. Source code, manual, binaries for 32 and 64-bit Linux systems and an installer for Microsoft Windows are provided. The core components of CoCoa are written in C++, while the graphical user interface is written in Java.

  13. Mapping sugar beet pectin acetylation pattern.

    Science.gov (United States)

    Ralet, Marie-Christine; Cabrera, Juan Carlos; Bonnin, Estelle; Quéméner, Bernard; Hellìn, Pilar; Thibault, Jean-François

    2005-08-01

    Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.

  14. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

    DEFF Research Database (Denmark)

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas

    2015-01-01

    or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue...... of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation...... functions as a protein repair factor that removes acetylation lesions from lysine residues....

  15. Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

    DEFF Research Database (Denmark)

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A

    2013-01-01

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells...... acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low...

  16. Purification and characterization of acetone carboxylase from Xanthobacter strain Py2

    OpenAIRE

    Sluis, Miriam K.; Ensign, Scott A.

    1997-01-01

    Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an α2β2γ2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis o...

  17. Ribulose 1,5-bisphosphate carboxylase synthesis during heat shock

    International Nuclear Information System (INIS)

    Vierling, E.; Key, J.L.

    1985-01-01

    Ribulose 1,5-bisphosphate carboxylase (RuBPCase) was chosen as a model protein to study how heat shock (HS) affects both chloroplast protein synthesis and the nuclear-chloroplast interaction in production of chloroplast proteins. Experiments were performed using highly chlorophyllous, soybean (Glycine max L. Merr. var Corsoy) cell suspension cultures active in chloroplast protein synthesis. Synthesis of RuBPCase large (L) and small (S) subunits was followed by in vivo labeling, and corresponding mRNA levels were examined by Northern and dot hybridization analyses. Results demonstrate that L and S synthesis declines with increasing HS temperatures (33-40 0 C) and reaches minimum levels (20-30% of control) at temperatures of maximum HS protein synthesis (39-40 0 C). Recovery of L and S synthesis following a 2-hour HS at 38 or 40 0 C was also studied. The changes in S synthesis during HS and recovery correlate with the steady state levels of S mRNA. In contrast, changes in L synthesis show little relationship to the corresponding mRNA levels; levels of L mRNA remain relatively unchanged by HS. The authors conclude that chloroplast protein synthesis shows no greater sensitivity to HS than is observed for cytoplasmic protein synthesis and that transport of proteins into the chloroplast (e.g.,S subunit) continues during HS. Furthermore, there is no apparent coordination of L and S subunit mNRA levels under the conditions examined

  18. Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis

    Directory of Open Access Journals (Sweden)

    F. Habarou

    2015-03-01

    Full Text Available Pyruvate carboxylase (PC is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A. Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1 and type B (patient 2. Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

  19. Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus.

    Science.gov (United States)

    Thorgersen, Michael P; Lipscomb, Gina L; Schut, Gerrit J; Kelly, Robert M; Adams, Michael W W

    2014-03-01

    The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO₂ and acetyl-CoA by the heterologous-expression of three enzymes from the CO₂ fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α₂β₂) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus. Copyright © 2014 International Metabolic Engineering Society. All rights reserved.

  20. Acetyl Phosphate as a Primordial Energy Currency at the Origin of Life

    Science.gov (United States)

    Whicher, Alexandra; Camprubi, Eloi; Pinna, Silvana; Herschy, Barry; Lane, Nick

    2018-03-01

    Metabolism is primed through the formation of thioesters via acetyl CoA and the phosphorylation of substrates by ATP. Prebiotic equivalents such as methyl thioacetate and acetyl phosphate have been proposed to catalyse analogous reactions at the origin of life, but their propensity to hydrolyse challenges this view. Here we show that acetyl phosphate (AcP) can be synthesised in water within minutes from thioacetate (but not methyl thioacetate) under ambient conditions. AcP is stable over hours, depending on temperature, pH and cation content, giving it an ideal poise between stability and reactivity. We show that AcP can phosphorylate nucleotide precursors such as ribose to ribose-5-phosphate and adenosine to adenosine monophosphate, at modest ( 2%) yield in water, and at a range of pH. AcP can also phosphorylate ADP to ATP in water over several hours at 50 °C. But AcP did not promote polymerization of either glycine or AMP. The amino group of glycine was preferentially acetylated by AcP, especially at alkaline pH, hindering the formation of polypeptides. AMP formed small stacks of up to 7 monomers, but these did not polymerise in the presence of AcP in aqueous solution. We conclude that AcP can phosphorylate biologically meaningful substrates in a manner analogous to ATP, promoting the origins of metabolism, but is unlikely to have driven polymerization of macromolecules such as polypeptides or RNA in free solution. This is consistent with the idea that a period of monomer (cofactor) catalysis preceded the emergence of polymeric enzymes or ribozymes at the origin of life.

  1. Regulation of schistosome egg production by HMG CoA reductase

    International Nuclear Information System (INIS)

    VandeWaa, E.A.; Bennett, J.L.

    1986-01-01

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of 14 C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production

  2. Unlocking the Barley Genome by Chromosomal and Comparative Genomics

    Czech Academy of Sciences Publication Activity Database

    Mayer, K. F. X.; Martis, M.; Hedley, P. E.; Šimková, Hana; Liu, H.; Morris, J. A.; Steuernagel, B.; Taudien, S.; Kubaláková, Marie; Suchánková, Pavla; Doležel, Jaroslav; Stein, N.

    2011-01-01

    Roč. 23, č. 4 (2011), s. 1249-1263 ISSN 1040-4651 Institutional research plan: CEZ:AV0Z50380511 Keywords : PSEUDO-RESPONSE-REGULATOR * ACETYL-COA CARBOXYLASE * TRITICUM-AESTIVUM L. Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.987, year: 2011

  3. The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis

    Science.gov (United States)

    van der Westhuyzen, Renier; Hammons, Justin C.; Meier, Jordan L.; Dahesh, Samira; Moolman, Wessel J. A.; Pelly, Stephen C.; Nizet, Victor; Burkart, Michael D.; Strauss, Erick

    2012-01-01

    SUMMARY The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics, and highlight CoA biosynthesis as a viable antimicrobial drug target. PMID:22633408

  4. Acetylation of woody lignocellulose: significance and regulation

    Directory of Open Access Journals (Sweden)

    Prashant Mohan-Anupama Pawar

    2013-05-01

    Full Text Available Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1 summarize literature on lignocellulose acetylation in different tree species, 2 present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3 describe plant proteins involved in lignocellulose O-acetylation, 4 give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5 discuss prospects for exploiting these enzymes in planta to modify xylan acetylation.

  5. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II

    Directory of Open Access Journals (Sweden)

    Andrew M. James

    2017-02-01

    Full Text Available Summary: Acetyl coenzyme A (AcCoA, a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3 reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2 can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. : James et al. show that the non-enzymatic N-acetylation of lysine residues in mitochondrial proteins frequently occurs via a proximal S-acetylated thiol intermediate. Glutathione equilibrates with this intermediate, allowing the thioesterase glyoxalase II to limit protein lysine N-acetylation. These findings expand our understanding of how protein acetylation arises. Keywords: AcetylCoA, lysine acetylation, glyoxalase

  6. Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

    International Nuclear Information System (INIS)

    Bame, K.J.; Rome, L.H.

    1985-01-01

    The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [ 3 H]CoA were found to produce acetyl-[ 3 H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [ 3 H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [ 3 H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate

  7. Investigation of acetyl migrations in furanosides

    Directory of Open Access Journals (Sweden)

    Migaud ME

    2006-07-01

    Full Text Available Abstract Standard reaction conditions for the desilylation of acetylated furanoside (riboside, arabinoside and xyloside derivatives facilitate acyl migration. Conditions which favour intramolecular and intermolecular mechanisms have been identified with intermolecular transesterifications taking place under mild basic conditions when intramolecular orthoester formations are disfavoured. In acetyl ribosides, acyl migration could be prevented when desilylation was catalysed by cerium ammonium nitrate.

  8. Mutations in COA3 cause isolated complex IV deficiency associated with neuropathy, exercise intolerance, obesity, and short stature.

    Science.gov (United States)

    Ostergaard, Elsebet; Weraarpachai, Woranontee; Ravn, Kirstine; Born, Alfred Peter; Jønson, Lars; Duno, Morten; Wibrand, Flemming; Shoubridge, Eric A; Vissing, John

    2015-03-01

    We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors. The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  9. Histone Acetylation in Fungal Pathogens of Plants

    Directory of Open Access Journals (Sweden)

    Junhyun Jeon

    2014-03-01

    Full Text Available Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed.

  10. Mode of action of Fusarium moniliforme endopolygalacturonase towards acetylated pectin.

    NARCIS (Netherlands)

    Bonnin, E.; Alebeek, van G.J.W.M.; Voragen, A.G.J.; Thibault, J.F.

    2003-01-01

    Endopolygalacturonase from Fusarium moniliforme was used to degrade acetylated homogalacturonan previously prepared from sugar beet pulp. The initial velocity and the final percentage of hydrolysis decreased-very rapidly with increasing degree of acetylation, showing that acetyl substitution

  11. Heat inactivation of leaf phosphoenolpyruvate carboxylase: Protection by aspartate and malate in C4 plants.

    Science.gov (United States)

    Rathnam, C K

    1978-01-01

    The activity of phosphoenolpyruvate (PEP) carboxylase EC 4.1.1.31 in leaf extracts of Eleusine indica L. Gaertn., a C4 plant, exhibited a temperature optimum of 35-37° C with a complete loss of activity at 50° C. However, the enzyme was protected effectively from heat inactivation up to 55° C by L-aspartate. Activation energies (Ea) for the enzyme in the presence of aspartate were 2.5 times lower than that of the control enzyme. Arrhenius plots of PEP carboxylase activity (±aspartate) showed a break in the slope around 17-20° C with a 3-fold increase in the Ea below the break. The discontinuity in the slopes was abolished by treating the enzyme extracts with Triton X-100, suggesting that PEP carboxylase in C4 plants is associated with lipid and may be a membrane bound enzyme. Depending upon the species, the major C4 acid formed during photosynthesis (malate or aspartate) was found to be more protective than the minor C4 acid against the heat inactivation of their PEP carboxylase. Oxaloacetate, the reaction product, was less effective compared to malate or aspartate. Several allosteric inhibitors of PEP carboxylase were found to be moderately to highly effective in protecting the C4 enzyme while its activators showed no significant effect. PEP carboxylase from C3 species was not protected from thermal inactivation by the C4 acids. The physiological significance of these results is discussed in relation to the high temperature tolerance of C4 plants.

  12. Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

    Science.gov (United States)

    Plaga, W; Frank, R; Knappe, J

    1988-12-15

    Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

  13. Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation.

    Directory of Open Access Journals (Sweden)

    Katja Merschbaecher

    Full Text Available Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs, and the antagonistic histone deacetylases (HDACs play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM. While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact of both, increased and decreased acetylation on formation of appetitive olfactory memory in honeybees. We show that learning-induced changes in the acetylation of histone H3 at aminoacid-positions H3K9 and H3K18 exhibit distinct and different dynamics depending on the training strength. A strong training that induces LTM leads to an immediate increase in acetylation at H3K18 that stays elevated for hours. A weak training, not sufficient to trigger LTM, causes an initial increase in acetylation at H3K18, followed by a strong reduction in acetylation at H3K18 below the control group level. Acetylation at position H3K9 is not affected by associative conditioning, indicating specific learning-induced actions on the acetylation machinery. Elevating acetylation levels by blocking HDACs after conditioning leads to an improved memory. While memory after strong training is enhanced for at least 2 days, the enhancement after weak training is restricted to 1 day. Reducing acetylation levels by blocking HAT activity after strong training leads to a suppression of transcription-dependent LTM. The memory suppression is also observed in case of weak training, which does not require transcription processes. Thus, our findings demonstrate that acetylation-mediated processes act as bidirectional regulators of memory formation that facilitate or suppress memory independent of its transcription-requirement.

  14. The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel

    NARCIS (Netherlands)

    Huberts, Daphne H. E. W.; Venselaar, Hanka; Vriend, Gert; Veenhuis, Marten; van der Klei, Ida J.

    Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenula polymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional

  15. A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters

    Directory of Open Access Journals (Sweden)

    Dominik M. Peter

    2016-04-01

    Full Text Available Coenzyme A (CoA is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives, and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives. Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.

  16. CoaSim: A Flexible Environment for Simulating Genetic Data under Coalescent Models

    DEFF Research Database (Denmark)

    Mailund; Schierup, Mikkel Heide; Pedersen, Christian Nørgaard Storm

    2005-01-01

    get insight into these. Results We have created the CoaSim application as a flexible environment for Monte various types of genetic data under equilibrium and non-equilibrium coalescent variety of applications. Interaction with the tool is through the Guile version scripting language. Scheme scripts......Background Coalescent simulations are playing a large role in interpreting large scale intra- polymorphism surveys and for planning and evaluating association studies. Coalescent of data sets under different models can be compared to the actual data to test different evolutionary factors and thus...

  17. Acetyl Fentanyl Toxicity: Two Case Reports.

    Science.gov (United States)

    Fort, Chelsea; Curtis, Byron; Nichols, Clay; Niblo, Cheryl

    2016-11-01

    Acetyl fentanyl is an illicit fentanyl analog recently appearing in forensic casework. A quantitative method was created for measuring acetyl fentanyl in various biological matrices acquired post-mortem due to recent positive screening results in casework. Initial detection by immunoassay and standard gas chromatography mass spectrometry (GC/MS) methods have been previously reported for acetyl fentanyl and are examined further here. A Selective Ion Monitoring (SIM) method was created using a GC/MS for quantitation. In two separate cases, acetyl fentanyl was found to be in similar concentrations to those previously reported and ruled to be the cause of death. Acetyl fentanyl concentrations were determined in blood samples, liver, brain, vitreous humor, and urine. Individual 1 had acetyl fentanyl concentrations as follows: heart blood-285 ng/mL, femoral blood-192 ng/mL, liver-1,100 ng/g, brain-620 ng/g, and urine-3,420 ng/mL. Individual 2 had acetyl fentanyl concentrations as follows: heart blood-210 ng/mL, femoral blood-255 ng/mL, urine-2,720 ng/mL and vitreous humor-140 ng/mL. Experimental conditions for screening and quantitation are provided, using immunoassay and GC/MS methods. Due to the recent emergence of acetyl fentanyl, more data will need to be generated to fully differentiate recreational and fatal concentrations of acetyl fentanyl to assist toxicologists accurately understanding its physiological impact. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Endophilin-A1 BAR domain interaction with arachidonyl CoA.

    Science.gov (United States)

    Petoukhov, Maxim V; Weissenhorn, Winfried; Svergun, Dmitri I

    2014-01-01

    Endophilin-A1 belongs to the family of BAR domain containing proteins that catalyze membrane remodeling processes via sensing, inducing and stabilizing membrane curvature. We show that the BAR domain of endophilin-A1 binds arachidonic acid and molds its coenzyme A (CoA) activated form, arachidonyl-CoA into a defined structure. We studied low resolution structures of endophilin-A1-BAR and its complex with arachidonyl-CoA in solution using synchrotron small-angle X-ray scattering (SAXS). The free endophilin-A1-BAR domain is shown to be dimeric at lower concentrations but builds tetramers and higher order complexes with increasing concentrations. Extensive titration SAXS studies revealed that the BAR domain produces a homogenous complex with the lipid micelles. The structural model of the complexes revealed two arachidonyl-CoA micelles bound to the distal arms of an endophilin-A1-BAR dimer. Intriguingly, the radius of the bound micelles significantly decreases compared to that of the free micelles, and this structural result may provide hints on the potential biological relevance of the endophilin-A1-BAR interaction with arachidonyl CoA.

  19. Flexible DAQ card for detector systems utilizing the CoaXPress communication standard

    International Nuclear Information System (INIS)

    Neue, G.; Hejtmánek, M.; Marčišovský, M.; Voleš, P.

    2015-01-01

    This work concerns the design and construction of a flexible FPGA based data acquisition system aimed for particle detectors. The interface card as presented was designed for large area detectors with millions of individual readout channels. Flexibility was achieved by partitioning the design into multiple PCBs, creating a set of modular blocks, allowing the creation of a wide variety of configurations by simply stacking functional PCBs together. This way the user can easily toggle the polarity of the high voltage bias supply or switch the downstream interface from CoaXPress to PCIe or stream directly HDMI. We addressed the issues of data throughput, data buffering, bias voltage generation, trigger timing and fine tuning of the whole readout chain enabling a smooth data transmission. On the current prototype, we have wire-bonded a MediPix2 MXR quad and connected it to a XILINX FPGA. For the downstream interface, we implemented the CoaXPress communication protocol, which enables us to stream data at 3.125 Gbps to a standard PC

  20. Insulin signaling regulates fatty acid catabolism at the level of CoA activation.

    Directory of Open Access Journals (Sweden)

    Xiaojun Xu

    2012-01-01

    Full Text Available The insulin/IGF signaling pathway is a highly conserved regulator of metabolism in flies and mammals, regulating multiple physiological functions including lipid metabolism. Although insulin signaling is known to regulate the activity of a number of enzymes in metabolic pathways, a comprehensive understanding of how the insulin signaling pathway regulates metabolic pathways is still lacking. Accepted knowledge suggests the key regulated step in triglyceride (TAG catabolism is the release of fatty acids from TAG via the action of lipases. We show here that an additional, important regulated step is the activation of fatty acids for beta-oxidation via Acyl Co-A synthetases (ACS. We identify pudgy as an ACS that is transcriptionally regulated by direct FOXO action in Drosophila. Increasing or reducing pudgy expression in vivo causes a decrease or increase in organismal TAG levels respectively, indicating that pudgy expression levels are important for proper lipid homeostasis. We show that multiple ACSs are also transcriptionally regulated by insulin signaling in mammalian cells. In sum, we identify fatty acid activation onto CoA as an important, regulated step in triglyceride catabolism, and we identify a mechanistic link through which insulin regulates lipid homeostasis.

  1. Breaking the ritual metabolic cycle in order to save acetyl CoA: A potential role for mitochondrial humanin in T2 bladder cancer aggressiveness

    Directory of Open Access Journals (Sweden)

    Nesreen Nabil Omar

    2017-06-01

    Full Text Available Introduction: Cancer cells may exhibit outsourcing of their high energy need in order to avoid the intrinsic mitochondrial apoptosis. Reduced mitochondrial respiration and accumulation of mitochondrial genome mutations are among metabolic transformations in this regard. Mitochondrial humanin (MT-RNR2 is a small peptide with anti-apoptotic activities attributed to binding some pro-apoptotic proteins. Aim of the work: The current study aims at investigating the expression of mitochondrial humanin in bladder tumor cells and the possible casting of humanin anti-apoptotic action through orchestrating some of the mitochondrial metabolic enzymes. Material and methods: Here messenger RNA of humanin, succinate dehydrogenase, glutaminase, isocitrate dehydrogenase were compared in tissues from patients with T2 bladder carcinoma in comparison to tumor associated normal tissues from the same patients. Levels of lactate and mitochondrial pyruvate carrier (MPC1 mRNA were determined to scrutinize the prevalence of aerobic glycolysis. Results: The present study found that tumor cells had suppressed aerobic glycolysis, augmented mitochondrial respiration and interrupted tricarboxylic acid cycle, all of which were suggested to serve tumor aggressiveness. MT-RNR2 was found closely related to the alterations in mitochondrial activity. Conclusion: MT-RNR2 plays its anti-apoptotic role partly by avoiding deploying energy from complete oxidation of organic compounds to inorganic wastes. Thus MT-RNR2 can potentially serve as a new biomarker in the diagnosis of bladder carcinoma especially that it is present in blood circulation.

  2. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

  3. Biochemical signatures mimicking multiple carboxylase deficiency in children with mutations in MT-ATP6.

    Science.gov (United States)

    Larson, Austin A; Balasubramaniam, Shanti; Christodoulou, John; Burrage, Lindsay C; Marom, Ronit; Graham, Brett H; Diaz, George A; Glamuzina, Emma; Hauser, Natalie; Heese, Bryce; Horvath, Gabriella; Mattman, Andre; van Karnebeek, Clara; Lane Rutledge, S; Williamson, Amy; Estrella, Lissette; Van Hove, Johan K L; Weisfeld-Adams, James D

    2018-01-04

    Elevations of specific acylcarnitines in blood reflect carboxylase deficiencies, and have utility in newborn screening for life-threatening organic acidemias and other inherited metabolic diseases. In this report, we describe a newly-identified association of biochemical features of multiple carboxylase deficiency in individuals harboring mitochondrial DNA (mtDNA) mutations in MT-ATP6 and in whom organic acidemias and multiple carboxylase deficiencies were excluded. Using retrospective chart review, we identified eleven individuals with abnormally elevated propionylcarnitine (C3) or hydroxyisovalerylcarnitine (C5OH) with mutations in MT-ATP6, most commonly m.8993T>G in high heteroplasmy or homoplasmy. Most patients were ascertained on newborn screening; most had normal enzymatic or molecular genetic testing to exclude biotinidase and holocarboxylase synthetase deficiencies. MT-ATP6 is associated with some cases of Leigh disease; clinical outcomes in our cohort ranged from death from neurodegenerative disease in early childhood to clinically and developmentally normal after several years of follow-up. These cases expand the biochemical phenotype associated with MT-ATP6 mutations, especially m.8993T>G, to include acylcarnitine abnormalities mimicking carboxylase deficiency states. Clinicians should be aware of this association and its implications for newborn screening, and consider mtDNA sequencing in patients exhibiting similar acylcarnitine abnormalities that are biotin-unresponsive and in whom other enzymatic deficiencies have been excluded. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  4. Phosphoenolpyruvate carboxylase from C4 leaves is selectively targeted for inhibition by anionic phospholipids

    NARCIS (Netherlands)

    Monreal, J.A.; McLoughlin, F.; Echevarría, C.; García-Mauriño, S.; Testerink, C.

    2010-01-01

    Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an enzyme playing a crucial role in photosynthesis of C4 plants. Here, we identify anionic phospholipids as novel regulators that inhibit C4 PEPC activity and provide evidence that the enzyme partially localizes to membranes.

  5. Characterization of phosphoenolpyruvate carboxylase from mature maize seeds: Properties of phosphorylated and dephosphorylated forms

    Czech Academy of Sciences Publication Activity Database

    Černý, M.; Doubnerová, V.; Müller, Karel; Ryšlavá, H.

    2010-01-01

    Roč. 92, č. 10 (2010), s. 1362-1370 ISSN 0300-9084 R&D Projects: GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50380511 Keywords : Phosphoenolpyruvate carboxylase * Phosphorylation * Seed Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.787, year: 2010

  6. Directing vanillin production from ferulic acid by increased acetyl-CoA consumption in recombinant Escherichia coli.

    Science.gov (United States)

    Lee, Eun-Gyeong; Yoon, Sang-Hwal; Das, Amitabha; Lee, Sook-Hee; Li, Cui; Kim, Jae-Yean; Choi, Myung-Suk; Oh, Deok-Kun; Kim, Seon-Won

    2009-01-01

    The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5alpha (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5alpha (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli.

  7. Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulase-bisphosphate carboxylase activity

    International Nuclear Information System (INIS)

    Berry, J.A.; Lorimer, G.H.; Pierce, J.; Seemann, J.R.; Meek, J.; Freas, S.

    1987-01-01

    The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-P 2 ) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] of leaves of Phaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-P 2 carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-P 2 carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-P 2 carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-P 2 carboxylase. The compound was analyzed by GC/MS, 13 C NMR, and 1 H NMR and shown to be 2-carboxyarabinitol 1-phosphate [(2-C-phosphohydroxymethyl)-D-ribonic acid]. The structure of the isolated compound differs from the Rbu-1,5-P 2 carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate compared with the bisphosphate. The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-P 2 carboxylase activity in vivo

  8. p53 Acetylation: Regulation and Consequences

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  9. p53 Acetylation: Regulation and Consequences

    International Nuclear Information System (INIS)

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer

  10. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    Energy Technology Data Exchange (ETDEWEB)

    Wubben, T.; Mesecar, A.D. (Purdue); (UIC)

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  11. Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

    DEFF Research Database (Denmark)

    Biely, Peter; Cziszarava, Maria; Agger, Jane W.

    2014-01-01

    Results The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most promin...

  12. Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.

    Directory of Open Access Journals (Sweden)

    Misty L Kuhn

    Full Text Available The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

  13. Reaction of phosphoenolpyruvate carboxylase with (Z)-3-bromophosphoenolpyruvate and (Z)-3-fluorophosphoenolpyruvate

    International Nuclear Information System (INIS)

    Diaz, E.; O'Laughlin, J.T.; O'Leary, M.H.

    1988-01-01

    (Z)-3-Bromophosphoenolpyruvate inactivates phosphoenolpyruvate carboxylase from maize in the presence of HCO 3 - and either Mg 2+ or Mn 2+ . The inactivation rate follows saturation kinetics. Inactivation is slower in the presence of phospholactate or epoxymaleate, both of which are inhibitors of the enzyme, or dithiothreitol. Inactivation is completely prevented by the presence of lactate dehydrogenase and NADH, and 3-bromolactate is formed during this treatment. If the reaction is conducted by using HC 18 O 3 - , the inorganic phosphate produced contains 18 O. This and other evidence indicate that phosphoenolpyruvate carboxylase catalyzes conversion of bromophosphoenolpyruvate into bromopyruvate by way of the usual carboxyphosphate-enolate intermediate, and bromopyruvate is the species responsible for enzyme inactivation. (Z)-3-fluorophosphoenolpyruvate is transformed by the enzyme into a 6:1 mixture of 3-fluoropyruvate and 3-fluorooxalacetate, presumably by the same mechanism. The enzyme is not inactivated during this treatment

  14. Expression, purification and crystallization of an archaeal-type phosphoenolpyruvate carboxylase

    International Nuclear Information System (INIS)

    Dharmarajan, Lakshmi; Kraszewski, Jessica L.; Mukhopadhyay, Biswarup; Dunten, Pete W.

    2009-01-01

    The expression, purification, crystallization and preliminary diffraction analysis of an archaeal-type phosphoenolpyruvate carboxylase are described. Complete highly redundant X-ray data have been measured from a crystal diffracting to 3.13 Å resolution. An archaeal-type phosphoenolpyruvate carboxylase (PepcA) from Clostridium perfringens has been expressed in Escherichia coli in a soluble form with an amino-terminal His tag. The recombinant protein is enzymatically active and two crystal forms have been obtained. Complete diffraction data extending to 3.13 Å resolution have been measured from a crystal soaked in KAu(CN) 2 , using radiation at a wavelength just above the Au L III edge. The asymmetric unit contains two tetramers of PepcA

  15. Genome sequence of carboxylesterase, carboxylase and xylose isomerase producing alkaliphilic haloarchaeon Haloterrigena turkmenica WANU15

    Directory of Open Access Journals (Sweden)

    Samy Selim

    2016-03-01

    Full Text Available We report draft genome sequence of Haloterrigena turkmenica strain WANU15, isolated from Soda Lake. The draft genome size is 2,950,899 bp with a G + C content of 64% and contains 49 RNA sequence. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LKCV00000000. Keywords: Soda Lake, Haloterrigena turkmenica, Carboxylesterase, Carboxylase, Xylose isomerase, Whole genome sequencing

  16. Purification and Characterization of the Acetone Carboxylase of Cupriavidus metallidurans Strain CH34

    Science.gov (United States)

    Rosier, Caroline; Leys, Natalie; Henoumont, Céline; Mergeay, Max

    2012-01-01

    Acetone carboxylase (Acx) is a key enzyme involved in the biodegradation of acetone by bacteria. Except for the Helicobacteraceae family, genome analyses revealed that bacteria that possess an Acx, such as Cupriavidus metallidurans strain CH34, are associated with soil. The Acx of CH34 forms the heterohexameric complex α2β2γ2 and can carboxylate only acetone and 2-butanone in an ATP-dependent reaction to acetoacetate and 3-keto-2-methylbutyrate, respectively. PMID:22492439

  17. Light-stimulated accumulation of transcripts of nuclear and chloroplast genes for ribulosebisphosphate carboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S M; Ellis, R J

    1981-01-01

    The chloroplast enzyme, ribulosebisphosphate carboxylase, consists of large subunit polypeptides encoded in the chloroplast genome and small subunit polypeptides encoded in the nuclear genome. Cloned DNA complementary to the small subunit mRNA hybridizes to a single RNA species of 900-1000 nucleotides in both total and poly(A)-containing RNA from leaves of Pisum sativum, but does not hybridize to chloroplast RNA. Small subunit cDNA hybridizes to at least three RNA species from nuclei, two of which are of higher molecular weight than the mature mRNA. A cloned large subunit DNA sequence hybridizes to a single species of Pisum chloroplast RNA containing approximately 1700 nucleotides, but does not hybridize to nuclear RNA. The light-stimulation of carboxylase accumulation reflects increases in the amounts of transcripts for both subunits in total leaf RNA. Transcripts of the small subunit gene are more abundant in nuclear RNA from light-grown leaves than in that from dark-grown leaves. These results suggest that the stimulation of carboxylase accumulation by light is mediated at the level of either transcription or RNA turnover in both nucleus and chloroplast.

  18. Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

    Science.gov (United States)

    Sun, Yingli; Du, Fengxia

    2017-01-01

    The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

  19. Production of Nα-acetyl Tα1-HSA through in vitro acetylation by RimJ.

    Science.gov (United States)

    Chen, Jing; Li, Haibin; Wang, Tao; Sun, Shuyang; Liu, Jia; Chen, Jianhua

    2017-11-10

    Thymosin alpha 1 (Tα1) is an important immunomodulating agent with various clinical applications. The natural form of Tα1 is N α -acetylated, which was supposed to be related to in vivo stability of the hormone. In this study, fusion protein Tα1-HSA was constructed and expressed in Pichia pastoris . RimJ, a N α -acetyltransferase from E.coli , was also overexpressed and purified to homogeneity. In vitro acetylation of Tα1-HSA in the presence of RimJ and acetyl coenzyme A resulted in N α -acetyl Tα1-HSA. The N α -acetylation was determined by LC-MS/MS. Kinetic assay indicated that RimJ had a higher affinity to desacetyl Tα1 than to Tα1-HSA. Bioactivity assay revealed fully retained activity of Tα1 when the hormone was connected to the N-terminus of the fusion protein, while the activity was compromised in our previously constructed HSA-Tα1. With fully retained activity and N-terminal acetylation, N α -acetyl Tα1-HSA was expected to be a more promising pharmaceutical agent than Tα1.

  20. Non-enzymatic N -acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S -acetylated Thiol Intermediate Sensitive to Glyoxalase II

    OpenAIRE

    James, Andrew M.; Hoogewijs, Kurt; Logan, Angela; Hall, Andrew R.; Ding, Shujing; Fearnley, Ian M.; Murphy, Michael P.

    2017-01-01

    Summary: Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysin...

  1. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II.

    Science.gov (United States)

    James, Andrew M; Hoogewijs, Kurt; Logan, Angela; Hall, Andrew R; Ding, Shujing; Fearnley, Ian M; Murphy, Michael P

    2017-02-28

    Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2) can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Liberdade e coação no direito de Kant

    Directory of Open Access Journals (Sweden)

    Pinheiro, Celso de Moraes

    2007-01-01

    Full Text Available Kant divide a filosofia moral em duas partes: Ética e Teoria da Justiça. Cada uma é compota de diferentes descrições de deveres e direitos. A ética contém deveres e direitos internos, voluntários e não-coercitivos. A teoria da justiça contém deveres e direitos externos e coercitivos. Os dois tipos de deveres e direitos são definidos em sua relação um com o outro. O que distingue os deveres éticos, ou deveres de virtude, dos deveres jurídicos, é que a compulsão externa para o dever de virtude é baseado na livre coerção própria. Assim, a finalidade deste artigo é pesquisar a noção de dever, e a relação entre dever, liberdade e coação

  3. Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers[W

    Science.gov (United States)

    Klempien, Antje; Kaminaga, Yasuhisa; Qualley, Anthony; Nagegowda, Dinesh A.; Widhalm, Joshua R.; Orlova, Irina; Shasany, Ajit Kumar; Taguchi, Goro; Kish, Christine M.; Cooper, Bruce R.; D’Auria, John C.; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

    2012-01-01

    Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway. PMID:22649270

  4. Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

    Science.gov (United States)

    Klempien, Antje; Kaminaga, Yasuhisa; Qualley, Anthony; Nagegowda, Dinesh A; Widhalm, Joshua R; Orlova, Irina; Shasany, Ajit Kumar; Taguchi, Goro; Kish, Christine M; Cooper, Bruce R; D'Auria, John C; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

    2012-05-01

    Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway.

  5. Adhesives for Achieving Durable Bonds with Acetylated Wood

    Science.gov (United States)

    Charles Frihart; Rishawn Brandon; James Beecher; Rebecca Ibach

    2017-01-01

    Acetylation of wood imparts moisture durability, decay resistance, and dimensional stability to wood; however, making durable adhesive bonds with acetylated wood can be more difficult than with unmodified wood. The usual explanation is that the acetylated surface has fewer hydroxyl groups, resulting in a harder-to-wet surface and in fewer hydrogen bonds between wood...

  6. Effect of Drying Pretreatment on the Acetylation of Nanofibrillated Cellulose

    Directory of Open Access Journals (Sweden)

    Vesna Zepič

    2015-10-01

    Full Text Available The aim of this study was to evaluate the effect of different morphologies of solvent-exchanged (NFCSE, spray-dried (NFCSD, and freeze-dried (NFCFD nano-fibrillated cellulose on the susceptibility to surface modification with the acetic anhydride/pyridine system. The degree of substitution (DS, morphology, degree of crystallinity (Icr, hydrophobicity, and thermal stability of acetylated products were examined. Acetylated NFCSD and NFCFD had higher DS than acetylated NFCSE, suggesting that drying pre-treatment increased the susceptibility of NFC for acetylation. The morphology of acetylated NFCFD and NFCSD with higher DS was different from unmodified samples, while that of NFCSE was not affected by acetylation. Microspheres of acetylated NFCSD started to dissolve when the highest DS was reached. As opposed to unmodified NFCFD, the nanofibrillar units of acetylated NFCFD became individualised at lower DS. Acetylated samples had lower Icr than the unmodified samples. A significant increase in the contact angle was observed at higher DS of acetylated NFC samples. Acetylation markedly elevated the thermal stability of the acetylated NFC samples.

  7. Abundance and distribution of archaeal acetyl-CoA/propionyl-CoA carboxylase genes indicative for putatively chemoautotrophic Archaea in the tropical Atlantic's interior

    NARCIS (Netherlands)

    Bergauer, K.; Sintes, E.; van Bleijswijk, J.; Witte, H.; Herndl, G.J.

    2013-01-01

    Recently, evidence suggests that dark CO2 fixation in the pelagic realm of the ocean does not only occur in the suboxic and anoxic water bodies but also in the oxygenated meso- and bathypelagic waters of the North Atlantic. To elucidate the significance and phylogeny of the key organisms mediating

  8. Phylogeny and evolutionary history of Leymus (Triticeae; Poaceae based on a single-copy nuclear gene encoding plastid acetyl-CoA carboxylase

    Directory of Open Access Journals (Sweden)

    Ding Cun-Bang

    2009-10-01

    Full Text Available Abstract Background Single- and low- copy genes are less likely subject to concerted evolution, thus making themselves ideal tools for studying the origin and evolution of polyploid taxa. Leymus is a polyploid genus with a diverse array of morphology, ecology and distribution in Triticeae. The genomic constitution of Leymus was assigned as NsXm, where Ns was presumed to be originated from Psathyrostachys, while Xm represented a genome of unknown origin. In addition, little is known about the evolutionary history of Leymus. Here, we investigate the phylogenetic relationship, genome donor, and evolutionary history of Leymus based on a single-copy nuclear Acc1 gene. Results Two homoeologues of the Acc1 gene were isolated from nearly all the sampled Leymus species using allele-specific primer and were analyzed with those from 35 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1 Leymus is closely related to Psathyrostachys, Agropyron, and Eremopyrum; (2 Psathyrostachys juncea is an ancestral Ns-genome donor of Leymus species; (3 the Xm genome in Leymus may be originated from an ancestral lineage of Agropyron and Eremopyrum triticeum; (4 the Acc1 sequences of Leymus species from the Qinghai-Tibetan plateau are evolutionarily distinct; (5 North America Leymus species might originate from colonization via the Bering land bridge; (6 Leymus originated about 11-12MYA in Eurasia, and adaptive radiation might have occurred in Leymus during the period of 3.7-4.3 MYA and 1.7-2.1 MYA. Conclusion Leymus species have allopolyploid origin. It is hypothesized that the adaptive radiation of Leymus species might have been triggered by the recent upliftings of the Qinghai-Tibetan plateau and subsequent climatic oscillations. Adaptive radiation may have promoted the rapid speciation, as well as the fixation of unique morphological characters in Leymus. Our results shed new light on our understanding of the origin of Xm genome, the polyploidization events and evolutionary history of Leymus that could account for the rich diversity and ecological adaptation of Leymus species.

  9. The glossyhead1 allele of acc1 reveals a principal role for multidomain acetyl-coenzyme a carboxylase in the biosynthesis of cuticular waxes by Arabidopsis

    KAUST Repository

    Lu, Shiyou; Zhao, Huayan; Parsons, Eugene P.; Xu, Changcheng; Kosma, Dylan K.; Xu, Xiaojing; Chao, Daiyin; Lohrey, Gregory T.; Bangarusamy, Dhinoth Kumar; Wang, Guangchao; Bressan, Ray Anthony; Jenks, Matthew A.

    2011-01-01

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1

  10. The growing landscape of lysine acetylation links metabolism and cell signalling

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Weinert, Brian Tate; Nishida, Yuya

    2014-01-01

    Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation...

  11. Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    Science.gov (United States)

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Khan, Bashir M

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Molecular analysis of virulent genes (coa and spa) of staphylococcus aureus involved in natural cases of bovine mastitis

    International Nuclear Information System (INIS)

    Khan, A.; Javed, M.T.; Mahmood, F.; Hussain, R.

    2013-01-01

    The present study was undertaken to determine the distribution and genotypic characteristics of Staphylococcus aureus isolates recovered from naturally occurring mastitis in cattle and buffaloes. For this purpose a total of 1445 lactating cattle (653) and buffaloes (792) present at two experimental livestock farms Okara (Bahadarnagar) and Sahiwal (Qadiarabad), in and around district Faisalabad and slaughtered at an abattoir due to low milk yield and were screened for mastitis. California Mastitis Test (CMT) was used to detect sub clinical mastitis. The positive quarter milk samples were collected for culturing of S. aureus isolates. taphylococcus aureus isolates were identified on the basis of growth features, biochemical characteristics, coagulase test and as well as amplification of coagulase (coa) and spa (spa-X) genes specific to its virulence. S. aureus isolates (n=265) were characterized by Polymerase chain reaction to determine the frequency of coagulase (coa) and spa (spa-X) genes. From these isolates the amplification of the coagulase (coa) gene yielded three different PCR products approximately 204bp to 490bp while spa (spa-X) gene produced five different products ranging in size from 190bp to 320bp. PCR revealed that from all the coagulase positive S. aureus isolates 261(98.5%) had spa (spa-X) gene. The results of the present study indicated that S. aureus isolates recovered from bovine mastitis were genetically different within and among the various herds which may provide essential and valuable strategies to control staphylococcal infections in future. (author)

  13. Molecular analysis of virulent genes (coa and spa) of staphylococcus aureus involved in natural cases of bovine mastitis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, A.; Javed, M. T.; Mahmood, F. [University of Agriculture, Faisalabad (Pakistan). Dept. of Pathology; Hussain, R. [The Islamia Univ. of Bahawalpur, Pakistan (Pakistan). Dept. of Veterinary and Animal Sciences

    2013-12-15

    The present study was undertaken to determine the distribution and genotypic characteristics of Staphylococcus aureus isolates recovered from naturally occurring mastitis in cattle and buffaloes. For this purpose a total of 1445 lactating cattle (653) and buffaloes (792) present at two experimental livestock farms Okara (Bahadarnagar) and Sahiwal (Qadiarabad), in and around district Faisalabad and slaughtered at an abattoir due to low milk yield and were screened for mastitis. California Mastitis Test (CMT) was used to detect sub clinical mastitis. The positive quarter milk samples were collected for culturing of S. aureus isolates. taphylococcus aureus isolates were identified on the basis of growth features, biochemical characteristics, coagulase test and as well as amplification of coagulase (coa) and spa (spa-X) genes specific to its virulence. S. aureus isolates (n=265) were characterized by Polymerase chain reaction to determine the frequency of coagulase (coa) and spa (spa-X) genes. From these isolates the amplification of the coagulase (coa) gene yielded three different PCR products approximately 204bp to 490bp while spa (spa-X) gene produced five different products ranging in size from 190bp to 320bp. PCR revealed that from all the coagulase positive S. aureus isolates 261(98.5%) had spa (spa-X) gene. The results of the present study indicated that S. aureus isolates recovered from bovine mastitis were genetically different within and among the various herds which may provide essential and valuable strategies to control staphylococcal infections in future. (author)

  14. Histone Acetylation Modifications Affect Tissue-Dependent Expression of Poplar Homologs of C4 Photosynthetic Enzyme Genes

    Directory of Open Access Journals (Sweden)

    Yuan Li

    2017-06-01

    Full Text Available Histone modifications play important roles in regulating the expression of C4 photosynthetic genes. Given that all enzymes required for the C4 photosynthesis pathway are present in C3 plants, it has been hypothesized that this expression regulatory mechanism has been conserved. However, the relationship between histone modification and the expression of homologs of C4 photosynthetic enzyme genes has not been well determined in C3 plants. In the present study, we cloned nine hybrid poplar (Populus simonii × Populus nigra homologs of maize (Zea mays C4 photosynthetic enzyme genes, carbonic anhydrase (CA, pyruvate orthophosphate dikinase (PPDK, phosphoenolpyruvate carboxykinase (PCK, and phosphoenolpyruvate carboxylase (PEPC, and investigated the correlation between the expression levels of these genes and the levels of promoter histone acetylation modifications in four vegetative tissues. We found that poplar homologs of C4 homologous genes had tissue-dependent expression patterns that were mostly well-correlated with the level of histone acetylation modification (H3K9ac and H4K5ac determined by chromatin immunoprecipitation assays. Treatment with the histone deacetylase inhibitor trichostatin A further confirmed the role of histone acetylation in the regulation of the nine target genes. Collectively, these results suggest that both H3K9ac and H4K5ac positively regulate the tissue-dependent expression pattern of the PsnCAs, PsnPPDKs, PsnPCKs, and PsnPEPCs genes and that this regulatory mechanism seems to be conserved among the C3 and C4 species. Our findings provide new insight that will aid efforts to modify the expression pattern of these homologs of C4 genes to engineer C4 plants from C3 plants.

  15. Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans

    NARCIS (Netherlands)

    Pouvreau, L.A.M.; Jonathan, M.C.; Kabel, M.A.; Hinz, S.W.A.; Gruppen, H.; Schols, H.A.

    2011-01-01

    Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated

  16. NetAcet: prediction of N-terminal acetylation sites

    DEFF Research Database (Denmark)

    Kiemer, Lars; Bendtsen, Jannick Dyrløv; Blom, Nikolaj

    2005-01-01

    Summary: We present here a neural network based method for prediction of N-terminal acetylation-by far the most abundant post-translational modification in eukaryotes. The method was developed on a yeast dataset for N-acetyltransferase A (NatA) acetylation, which is the type of N-acetylation for ......Summary: We present here a neural network based method for prediction of N-terminal acetylation-by far the most abundant post-translational modification in eukaryotes. The method was developed on a yeast dataset for N-acetyltransferase A (NatA) acetylation, which is the type of N...

  17. Acetylation and characterization of banana (Musa paradisiaca) starch.

    Science.gov (United States)

    Bello-Pérez, L A; Contreras-Ramos, S M; Jìmenez-Aparicio, A; Paredes-López, O

    2000-01-01

    Banana native starch was acetylated and some of its functional properties were evaluated and compared to corn starch. In general, acetylated banana starch presented higher values in ash, protein and fat than corn acetylated starch. The modified starches had minor tendency to retrogradation assessed as % transmittance of starch pastes. At high temperature acetylated starches presented a water retention capacity similar to their native counterpart. The acetylation considerably increased the solubility of starches, and a similar behavior was found for swelling power. When freeze-thaw stability was studied, acetyl banana starch drained approximately 60% of water in the first and second cycles, but in the third and fourth cycles the percentage of separated water was low. However, acetyl corn starch showed lower freeze-thaw stability than the untreated sample. The modification increased the viscosity of banana starch pastes.

  18. Fragrance material review on acetyl carene.

    Science.gov (United States)

    Scognamiglio, J; Letizia, C S; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl carene when used as a fragrance ingredient is presented. Acetyl carene is a member of the fragrance structural group Alkyl Cyclic Ketones. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl carene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, and skin sensitization data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013A Toxicologic and dermatologic assessment of alkyl cyclic ketones when used as fragrance ingredients. (submitted for publication).) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. Copyright © 2013. Published by Elsevier Ltd.

  19. Immunohistochemical expression of alpha methylacyl-coa racemase (amacr) in carcinoma prostate in pakistani population

    International Nuclear Information System (INIS)

    Tariq, H.; Ahmed, R.; Muhammad, I.; Afzal, M.S.; Hashmi, S.N.; Hamdani, S.N.R.; Shahid, A.

    2017-01-01

    Objective: To determine the frequency of expression of positive diagnostic marker alpha methylacyl-COA RACEMES (AMACR) in the examination of prostate needle biopsy specimens from patients of adenocarcinoma prostate from a subset of Pakistani population. Study design: Cross-sectional study. Place and Duration of Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi from Apr 2015 to Oct 2015. Material and Methods: All specimens of adenocarcinoma prostate diagnosed at Armed forces institute of pathology on the basis of immunohistochemistry and routine histopathology irrespective of age of patient, histological type or grade of the tumor were analyzed. Mean and Standard deviation were calculated for quantitative variables like patient's age and frequencies along with percentages were calculated for qualitative variables like AMACR expression. Results: Out of the total 80 cases, 68 (85%) were positive for AMACR while 12 (15%) were negative. Among the cases that were negative 9 (11.3%) showed 1 +- staining (Weak, non-circumferential) and 3 cases (3.8%) displayed 0 staining (No cytoplasmic staining). Conclusion: Positive staining for AMACR can be used to support a diagnosis of cancer on prostate needle core biopsies when the focus in question is <1mm in maximum dimension. The results of AMACR expression in a subset of Pakistani population are comparable to the western studies. AMACR staining must be interpreted in the context of basic haematoxylin and eosin criteria for malignancy along with the results expansion of other supportive markers, such as a basal cell specific marker like p63 or 34 beta E12. (author)

  20. Intracellular long-chain acyl CoAs activate TRPV1 channels.

    Directory of Open Access Journals (Sweden)

    Yi Yu

    Full Text Available TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs, another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP2-binding residues in TRPV1. In contrast to PIP2, LC-CoA modulation of TRPV1 is independent of Ca2+i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca2+i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.

  1. Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoAcarboxylase, temperature, and co-immobilization with Azospirillum brasilense [corrected].

    Science.gov (United States)

    Leyva, Luis A; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  2. The development of a decision analytic model of changes in mean deviation in people with glaucoma: the COA model.

    Science.gov (United States)

    Kymes, Steven M; Lambert, Dennis L; Lee, Paul P; Musch, David C; Siegfried, Carla J; Kotak, Sameer V; Stwalley, Dustin L; Fain, Joel; Johnson, Chris; Gordon, Mae O

    2012-07-01

    To create and validate a statistical model predicting progression of primary open-angle glaucoma (POAG) assessed by loss of visual field as measured in mean deviation (MD) using 3 landmark studies of glaucoma progression and treatment. A Markov decision analytic model using patient level data described longitudinal MD changes over 7 years. Patient-level data from the Collaborative Initial Glaucoma Treatment Study (n = 607), the Ocular Hypertension Treatment Study (OHTS; n = 148; only those who developed POAG in the first 5 years of OHTS) and Advanced Glaucoma Intervention Study (n = 591), the COA model. We developed a Markov model with transition matrices stratified by current MD, age, race, and intraocular pressure categories and used a microsimulation approach to estimate change in MD over 7 years. Internal validation compared model prediction for 7 years to actual MD for COA participants. External validation used a cohort of glaucoma patients drawn from university clinical practices. Change in visual field as measured in MD in decibels (dB). Regressing the actual MD against the predicted produced an R(2) of 0.68 for the right eye and 0.63 for the left. The model predicted ending MD for right eyes of 65% of participants and for 63% of left eyes within 3 dB of actual results at 7 years. In external validation the model had an R(2) of 0.79 in the right eye and 0.77 in the left at 5 years. The COA model is a validated tool for clinicians, patients, and health policy makers seeking to understand longitudinal changes in MD in people with glaucoma. Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  3. Fragrance material review on acetyl cedrene.

    Science.gov (United States)

    Scognamiglio, J; Letizia, C S; Politano, V T; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl cedrene when used as a fragrance ingredient is presented. Acetyl cedrene is a member of the fragrance structural group Alkyl Cyclic Ketones. The generic formula for this group can be represented as (R1)(R2)CO. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl cedrene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, and genotoxicity data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (2013) (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. Submitted with this manuscript.) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. Copyright © 2013. Published by Elsevier Ltd.

  4. Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism.

    NARCIS (Netherlands)

    Ettema, T.J.G.; Makarova, K.S.; Jellema, G.L.; Gierman, H.J.; Koonin, E.V.; Huynen, M.A.; Vos, W.M. de; Oost, J. van der

    2004-01-01

    Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized

  5. Identification and functional verification of Archaeal-type phosphoenolpyruvate carboxylase, a missing link in Archaeal central carbohydrate metabolism

    NARCIS (Netherlands)

    Ettema, T.J.G.; Makarova, K.S.; Jellema, G.L.; Gierman, H.J.; Koonin, E.V.; Huynen, M.A.; Vos, de W.M.; Oost, van der J.

    2004-01-01

    Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized

  6. Vitamin K-dependent carboxylase: Minimized escape of CO2 from solution may prolong linearity of the reaction rate

    International Nuclear Information System (INIS)

    Soute, B.A.; Bude, R.; Buitenhuis, H.; Vermeer, C.

    1989-01-01

    Escape of 14 CO 2 from the reaction mixture into the gas phase may seriously affect the accuracy of in vitro measurement of vitamin K-dependent carboxylase activity (and probably that of other carboxylases as well). In this paper we describe the effect of (a) the volume of the test tubes in which the reaction is performed, (b) the addition of an excess of NaH 12 CO 3 in parallel with standard amounts of NaH 14 CO 3 , and (c) the incubation temperature. In this way optimal conditions are defined and used for the carboxylation of various peptide and protein substrates. It is shown that both a prosequence and an internal recognition site contribute to the effective recognition of a substrate by carboxylase. The maximal efficiency of carboxylation was 1-2% with substrates lacking both signals and 20-50% if only one was present. This indicates the need for developing peptide substrates containing both recognition signals for vitamin K-dependent carboxylase

  7. Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia : Mollusca)

    NARCIS (Netherlands)

    Cavanaugh, Colleen M.; Abbott, Marilyn S.; Veenhuis, Marten

    1988-01-01

    The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP2Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large

  8. Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding

    NARCIS (Netherlands)

    Kirkeby, S.; Moe, D.; Bøg-Hansen, T. C.; van Noorden, C. J.

    1993-01-01

    Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding

  9. Differential patterns of histone acetylation in inflammatory bowel diseases

    Directory of Open Access Journals (Sweden)

    Adcock Ian M

    2011-01-01

    Full Text Available Abstract Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD to study the expression of acetylated histones (H 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP in dextran sulfate sodium (DSS-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.

  10. Structure, morphology and functionality of acetylated and oxidised barley starches.

    Science.gov (United States)

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Pinto, Vânia Zanella; Bartz, Josiane; Radunz, Marjana; Carreño, Neftali Lenin Villarreal; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2015-02-01

    Acetylation and oxidation are chemical modifications which alter the properties of starch. The degree of modification of acetylated and oxidized starches is dependent on the catalyst and active chlorine concentrations, respectively. The objective of this study was to evaluate the effect of acetylation and oxidation on the structural, morphological, physical-chemical, thermal and pasting properties of barley starch. Barley starches were acetylated at different catalyst levels (11%, 17%, and 23% of NaOH solution) and oxidized at different sodium hypochlorite concentrations (1.0%, 1.5%, and 2.0% of active chlorine). Fourier-transformed infrared spectroscopy (FTIR), X-ray diffractograms, thermal, morphological, and pasting properties, swelling power and solubility of starches were evaluated. The degree of substitution (DS) of the acetylated starches increased with the rise in catalyst concentration. The percentage of carbonyl (CO) and carboxyl (COOH) groups in oxidized starches also increased with the rise of active chlorine level. The presence of hydrophobic acetyl groups, carbonyl and carboxyl groups caused a partial disorganization and depolymerization of starch granules. The structural, morphological and functional changes in acetylated and oxidized starches varied according to reaction conditions. Acetylation makes barley starch more hydrophobic by the insertion of acetyl groups. Also the oxidation promotes low retrogradation and viscosity. All these characteristics are important for biodegradable film production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    DEFF Research Database (Denmark)

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double....... The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...... in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell...

  12. Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

    DEFF Research Database (Denmark)

    Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk

    2013-01-01

    We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

  13. Inhibition of E. coli P-enolpyruvate carboxylase by P-enol-3-bromopyruvate

    International Nuclear Information System (INIS)

    Asem, K.; Smith, T.E.

    1986-01-01

    The generality of the mechanism based inhibition of P-enolpyruvate carboxylases (PEPCase) by P-enol-3-bromopyruvate (BrPEP) was tested by measuring its effects on the allosterically regulated enzyme from E. coli. In the presence of 1mM Mn 2+ , BrPEP appears to be a competitive inhibitor (K/sub i/ = 0.0087mM) of PEPCase. Incubation of 0.005mM PEPCase with 0.5mM (or 1.0mM)BrPEP along with H 14 CO 3 - and Mn 2+ , yielded, upon reduction with NaBH 4 , a protein containing radioactivity in an amount approximately proportional to that expected from the loss of catalytic activity. At both a 25- and a 50-fold excess (0.5mM and 1.0mM, respectively) of BrPEP to PEPCase subunits, first order loss of activity occurred with k values of 5.24 x 10 -3 min -1 and 1.03 x 10 -2 min -1 , respectively. At the lower concentration of BrPEP the inactivation process appeared to be reversible after 40 min with no further inhibition occurring even up to two hours of incubation. At the higher concentration of BrPEP, the rate of inhibition slowed dramatically after 50 min and appeared insignificant over the next hour. These data suggest that BrPEP irreversibly inactivates the E. coli PEP carboxylase, but that there may be considerable dissociation of the product, Br-oxaloacetate, before irreversible binding occurs, and that the reduced rate of inactivation may be due to depletion of BrPEP

  14. Discovery and characterization of Ku acetylation in Mycobacterium smegmatis.

    Science.gov (United States)

    Zhou, Ying; Chen, Tao; Zhou, Lin; Fleming, Joy; Deng, Jiaoyu; Wang, Xude; Wang, Liwei; Wang, Yingying; Zhang, Xiaoli; Wei, Wenjing; Bi, Lijun

    2015-03-01

    Lysine acetylation is an important post-translational modification and is known to regulate many eukaryotic cellular processes. Little, however, is known about acetylated proteins in prokaryotes. Here, using immunoblotting, mass spectrometry and mutagenesis studies, we investigate the acetylation dynamics of the DNA repair protein Ku and its relationship with the deacetylase protein Sir2 and the non-homologous end joining (NHEJ) pathway in Mycobacterium smegmatis. We report that acetylation of Ku increases with growth, while NHEJ activity decreases, providing support for the hypothesis that acetylation of Ku may be involved in the DNA damage response in bacteria. Ku has multiple lysine sites. Our results indicate that K29 is an important acetylation site and that deficiency of Sir2 or mutation of K29 affects the quantity of Ku and its acetylation dynamics. Our findings expand knowledge of acetylation targets in prokaryotes and indicate a new direction for further research on bacterial DNA repair mechanisms. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Influence of acetylation on the physicochemical properties of ...

    African Journals Online (AJOL)

    The study investigates the effect of acetylation on the physicochemical properties of composited starches from sweet potato and water yam. Starch was respectively isolated from both sources, dried and subjected to acetylation at different combination. The result shows that the modified starches were of low percentage of ...

  16. The kinetics of the acetylation of gelatinised potato starch

    NARCIS (Netherlands)

    de Graaf, R.A.; Broekroelofs, G.A.; Janssen, L.P.B.M.; Beenackers, A.A C M

    1995-01-01

    The reaction rates, in the base-catalysed acetylation of gelatinised aqueous starch (4 wt%), by vinylacetate (ViAc), were investigated in a semibatch reactor at temperatures ranging from 20 to 50 degrees C. The desired starch acetylation reaction is accompanied by an undesired parallel

  17. Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

    2004-01-01

    textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a

  18. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Science.gov (United States)

    2010-04-01

    ... and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N-acetyl-L...

  19. Acetylated starch of Ofada rice as a sustained polymer in ...

    African Journals Online (AJOL)

    Objectives: To formulate and evaluate repaglinide microspheres using acetylated starch of the indigenous rice species Oryza glaberrima Steud (Ofada) as polymer. Materials and Methods: Ofada rice starch was acetylated with acetic anhydride in pyridine (DS 2.68) and characterized for morphology (Scanning electron ...

  20. Regulation of autophagy by cytosolic acetyl-coenzyme A

    DEFF Research Database (Denmark)

    Mariño, Guillermo; Pietrocola, Federico; Eisenberg, Tobias

    2014-01-01

    Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic p...

  1. Efficient acetylation of primary amines and amino acids in ...

    Indian Academy of Sciences (India)

    This effort represents the first efficient use of this most reactive but cheap acetylating agent to acetylate amines in excellent yields in aqueous medium. This is a potentially useful green chemical transformation where reaction takes place in environment-friendly brine solution leading to easy work-up and isolation of the ...

  2. The biology of lysine acetylation integrates transcriptional programming and metabolism

    Directory of Open Access Journals (Sweden)

    Mujtaba Shiraz

    2011-03-01

    Full Text Available Abstract The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT, there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

  3. Acetylation/deacetylation reactions of T-2, acetyl T-2, HT-2, and acetyl HT-2 toxins in bovine rumen fluid in vitro

    International Nuclear Information System (INIS)

    Munger, C.E.; Ivie, G.W.; Christopher, R.J.; Hammock, B.D.; Phillips, T.D.

    1987-01-01

    A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, underwent both acetylation and deacetylation reactions when incubated with bovine rumen fluid in vitro. Products from incubations of T-2 in rumen fluid included acetyl T-2, HT-2, and acetyl HT-2. Direct studies with tritiated samples of each of these metabolites confirmed their relatively facile interconversion in the rumen. Studies with [ 3 H]HT-2 under conditions of inhibited esterase activity (added diisopropyl fluorophosphate) showed that acetylation is preferred at C-3 vs. C-4. Studies with [ 3 H]acetyl T-2 indicated that deacetylation similarly occurs with greater rapidity at C-3. There were no indications that ester hydrolysis of these trichothecenes occurred at C-8 or C-15 or that they were subjected to epoxide reduction reactions. These data suggest that acetylation of T-2 and other trichothecenes in the rumen in situ may ultimately result in the absorption of more lipophilic metabolites whose toxicological and residual properties are at present unknown

  4. Acetylation/deacetylation reactions of T-2, acetyl T-2, HT-2, and acetyl HT-2 toxins in bovine rumen fluid in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Munger, C.E.; Ivie, G.W.; Christopher, R.J.; Hammock, B.D.; Phillips, T.D.

    A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, underwent both acetylation and deacetylation reactions when incubated with bovine rumen fluid in vitro. Products from incubations of T-2 in rumen fluid included acetyl T-2, HT-2, and acetyl HT-2. Direct studies with tritiated samples of each of these metabolites confirmed their relatively facile interconversion in the rumen. Studies with (/sup 3/H)HT-2 under conditions of inhibited esterase activity (added diisopropyl fluorophosphate) showed that acetylation is preferred at C-3 vs. C-4. Studies with (/sup 3/H)acetyl T-2 indicated that deacetylation similarly occurs with greater rapidity at C-3. There were no indications that ester hydrolysis of these trichothecenes occurred at C-8 or C-15 or that they were subjected to epoxide reduction reactions. These data suggest that acetylation of T-2 and other trichothecenes in the rumen in situ may ultimately result in the absorption of more lipophilic metabolites whose toxicological and residual properties are at present unknown.

  5. [A novel gene (Aa-accA ) encoding acetyl-CoA carboxyltransferase alpha-subunit of Alkalimonas amylolytica N10 enhances salt and alkali tolerance of Escherichia coli and tobacco BY-2 cells].

    Science.gov (United States)

    Xian, Mingjie; Zhai, Lei; Zhong, Naiqin; Ma, Yiwei; Xue, Yanfen; Ma, Yanhe

    2013-08-04

    Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid synthesis. In most bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Of them, accA encodes acetyl-CoA carboxyltransferase alpha-subunit. Our prior work on proteomics of Alkalimonas amylolytica N10 showed that the expression of the Aa-accA has a remarkable response to salt and alkali stress. This research aimed to find out the Aa-accA gene contributing to salt and alkali tolerance. The Aa-accA was amplified by PCR from A. amylolytica N10 and expressed in E. coli K12 host. The effects of Aa-accA expression on the growth of transgenic strains were examined under different NaCl concentration and pH conditions. Transgenic tobacco BY-2 cells harboring Aa-accA were also generated via Agrobacterium-mediated transformation. The viability of BY-2 cells was determined with FDA staining method after salt and alkali shock. The Aa-accA gene product has 318 amino acids and is homologous to the carboxyl transferase domain of acyl-CoA carboxylases. It showed 76% identity with AccA (acetyl-CoA carboxylase carboxyltransferase subunit alpha) from E. coli. Compared to the wild-type strains, transgenic E. coli K12 strain containing Aa-accA showed remarkable growth superiority when grown in increased NaCl concentrations and pH levels. The final cell density of the transgenic strains was 2.6 and 3.5 times higher than that of the control type when they were cultivated in LB medium containing 6% (W/V) NaCl and at pH 9, respectively. Complementary expression of Aa-accA in an accA-depletion E. coli can recover the tolerance of K12 delta accA to salt and alkali stresses to some extent. Similar to the transgenic E. coli, transgenic tobacco BY-2 cells showed higher percentages of viability compared to the wild BY-2 cells under the salt or alkali stress condition. We found that Aa-accA from A. amylolytica N10 overexpression enhances the tolerance of both transgenic E. coli and tobacco BY-2 cells to

  6. Studies on enzymes of C-4 pathway : Part V - Comparative studies of RUP2 carboxylase/oxygenase from maize and spinach

    International Nuclear Information System (INIS)

    Ramakrishna, J.; Bhagwat, A.S.; Sane, P.V.

    1978-01-01

    RuP 2 carboxylase (EC 4.1.1.39) isolated from maize, a C-4 plant possessed oxygenase activity. The ratio of carboxylase/oxygenase in the case of maize enzyme was more than 2-fold as compared to that of spinach. Fructose-1 6-diphosphate preferentially inhibited oxygenase function of the RuP 2 carboxylase/oxygenase in both the species when both the activities were assayed under identical conditions of pH, temperature, MgCl 2 , O 2 and RuP 2 concentration. Frutose-1, 6-diphosphate showed a fully competitive inhibition with respect to RuP 2 in the case of spinach, however the maize enzyme was inhibited seminoncompetitively. ( 14 C)-HCO 3 was used in the carboxylase assay. (author)

  7. Cobalt-vitamin B12 deficiency and the activity of methylmalonyl CoA mutase and methionine synthase in cattle.

    Science.gov (United States)

    Kennedy, D G; Young, P B; Kennedy, S; Scott, J M; Molloy, A M; Weir, D G; Price, J

    1995-01-01

    Cobalt deficiency was induced in cattle by feeding two groups of animals either a basal diet that was very low in Co (12.9-17.6 micrograms Co per kg), or the same diet supplemented with cobalt, for a total of 64 weeks. Vitamin B12 deficiency was induced, as judged by hepatic concentrations of vitamin B12 and plasma concentrations of MMA. However, the activity of holo-methylmalonyl CoA mutase was significantly reduced only in brain. This was reflected in very minor alterations in the tissue concentrations of branched chain- and odd numbered-fatty acids. The activity of holo-methionine synthase was significantly reduced in liver and brain, but there were no consequent alterations in the concentrations of phosphatidyl choline and phosphatidyl ethanolamine. This study confirms that cattle are less susceptible to the effects of cobalt deficiency than sheep, and concludes that prolonged cobalt deficiency had little significant effect on tissue metabolism.

  8. Diagnostic utility of alpha-methylacyl CoA racemase (P504S) on prostate needle biopsy.

    Science.gov (United States)

    Jiang, Zhong; Woda, Bruce A

    2004-11-01

    Alpha-methylacyl CoA racemase (AMACR), also known as P504S, was identified by the analysis of cDNA library subtraction in conjunction with high throughput microarray screening from prostate tissue and has been proven to be one of the very few biomarkers that can distinguish cancer from benign cells with high sensitivity and specificity for prostate carcinoma. It is a successful example of the translation of molecular findings into clinical practice. This review focuses on the study of AMACR (P504S) expression in small focal prostate cancer and atypical small acinar proliferation (ASAP) on needle biopsies and emphasizes the utility of AMACR (P504S) in routine surgical pathology practice. We also discuss the potential pitfalls and caveats in the interpretation of immunostaining results.

  9. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    DEFF Research Database (Denmark)

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian

    2015-01-01

    , and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide...... dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes....

  10. Fatty acid CoA ligase-4 gene polymorphism influences fatty acid metabolism in metabolic syndrome, but not in depression.

    Science.gov (United States)

    Zeman, Miroslav; Vecka, Marek; Jáchymová, Marie; Jirák, Roman; Tvrzická, Eva; Stanková, Barbora; Zák, Ales

    2009-04-01

    The composition of polyunsaturated fatty acids (PUFAs) in cell membranes and body tissues is altered in metabolic syndrome (MetS) and depressive disorder (DD). Within the cell, fatty acid coenzyme A (CoA) ligases (FACLs) activate PUFAs by esterifying with CoA. The FACL4 isoform prefers PUFAs (arachidonic and eicosapentaenoic acid) as substrates, and the FACL4 gene is mapped to Xq23. We have analyzed the association between the common single nucleotide polymorphism (SNP) (rs1324805, C to T substitution) in the first intron of the FACL4 gene and MetS or DD. The study included 113 healthy subjects (54 Males/59 Females), 56 MetS patients (34M/22F) and 41 DD patients (7M/34F). In MetS group, T-carriers and patients with CC or C0 (CC/C0) genotype did not differ in the values of metabolic indices of MetS and M/F ratio. Nevertheless, in comparison with CC/C0, the T-allele carriers were characterized by enhanced unfavorable changes in fatty acid metabolism typical for MetS: higher content of dihomogammalinolenic acid (P phosphatidylcholine (PC) (P = 0.052), lower index of Delta5 desaturation (P insulin, conjugated dienes and index of insulin resistance, but showed no significant association with the studied SNP. The present study shows that the common SNP (C to T substitution) in the first intron of the FACL4 gene is associated with altered FA composition of plasma phosphatidylcholines in patients with MetS.

  11. Up-regulation of hepatic Acyl CoA: Diacylglycerol acyltransferase-1 (DGAT-1) expression in nephrotic syndrome.

    Science.gov (United States)

    Vaziri, Nosratola D; Kim, Choong H; Phan, Dennis; Kim, Sara; Liang, Kaihui

    2004-07-01

    Nephrotic syndrome is associated with hypercholesterolemia, hypertriglyceridemia, and marked elevations of plasma low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Hypertriglyceridemia in nephrotic syndrome is accompanied by increased hepatic fatty acid synthesis, elevated triglyceride secretion, as well as lipoprotein lipase, VLDL-receptor, and hepatic triglyceride lipase deficiencies, which lead to impaired clearance of triglyceride-rich lipoproteins. Acyl CoA: diacylglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl CoA to 1, 2-diacylglycerol to form triglyceride. Two distinct DGATs (DGAT-1 and DGAT2) have recently been identified in the liver and other tissues. The present study tested the hypothesis that the reported increase in hepatic triglyceride secretion in nephrotic syndrome may be caused by up-regulation of DGAT. Male Sprague-Dawley rats were rendered nephrotic by two sequential injections of puromycin aminonucleoside (130 mg/kg on day 1 and 60 mg/kg on day 14) and studied on day 30. Placebo-treated rats served as controls. Hepatic DGAT-1 and DGAT-2 mRNA abundance and enzymatic activity were measured. The nephrotic group exhibited heavy proteinuria, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, and marked elevation of VLDL concentration. Hepatic DGAT-1 mRNA, DGAT-1, and total DGAT activity were significantly increased, whereas DGAT-2 mRNA abundance and activity were unchanged in the nephrotic rats compared to the control animals. The functional significance of elevation of DGAT activity was illustrated by the reduction in microsomal free fatty acid concentration in the liver of nephrotic animals. Nephrotic syndrome results in up-regulation of hepatic DGAT-1 expression and activity, which can potentially contribute to the associated hypertriglyceridemia by enhancing triglyceride synthesis. Thus, it appears that both depressed catabolism and increased synthetic capacity contribute to

  12. Nitrate-Dependent Degradation of Acetone by Alicycliphilus and Paracoccus Strains and Comparison of Acetone Carboxylase Enzymes ▿

    Science.gov (United States)

    Dullius, Carlos Henrique; Chen, Ching-Yuan; Schink, Bernhard

    2011-01-01

    A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601T by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria. PMID:21841031

  13. Acetone and Butanone Metabolism of the Denitrifying Bacterium “Aromatoleum aromaticum” Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

    Science.gov (United States)

    Schühle, Karola

    2012-01-01

    The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium “Aromatoleum aromaticum” is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)2 composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 Pi. The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content. PMID:22020645

  14. Histone acetylation regulates the time of replication origin firing.

    Science.gov (United States)

    Vogelauer, Maria; Rubbi, Liudmilla; Lucas, Isabelle; Brewer, Bonita J; Grunstein, Michael

    2002-11-01

    The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

  15. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    DEFF Research Database (Denmark)

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J

    2011-01-01

    Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

  16. Synthetic biology for engineering acetyl coenzyme a metabolism in yeast

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2014-01-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting...... chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl...

  17. Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity by the Activase System in Lysed Spinach Chloroplasts

    Science.gov (United States)

    Parry, Martin A. J.; Keys, Alfred J.; Foyer, Christine H.; Furbank, Robert T.; Walker, David A.

    1988-01-01

    Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO2 and Mg2+ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO2 and Mg2+. PMID:16666184

  18. Characterization of the mycobacterial acyl-CoA carboxylase holo complexes reveals their functional expansion into amino acid catabolism.

    Directory of Open Access Journals (Sweden)

    Matthias T Ehebauer

    2015-02-01

    Full Text Available Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.

  19. Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.

    Science.gov (United States)

    Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid; Band, Vimla

    2016-10-01

    Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Breast Cancer-Derived Lung Metastases Show Increased Pyruvate Carboxylase-Dependent Anaplerosis

    Directory of Open Access Journals (Sweden)

    Stefan Christen

    2016-10-01

    Full Text Available Cellular proliferation depends on refilling the tricarboxylic acid (TCA cycle to support biomass production (anaplerosis. The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis in breast-cancer-derived lung metastases compared to their primary cancers using in vivo 13C tracer analysis. We discovered that lung metastases have higher PC-dependent anaplerosis compared to primary breast cancers. Based on in vitro analysis and a mathematical model for the determination of compartment-specific metabolite concentrations, we found that mitochondrial pyruvate concentrations can promote PC-dependent anaplerosis via enzyme kinetics. In conclusion, we show that breast cancer cells proliferating as lung metastases activate PC-dependent anaplerosis in response to the lung microenvironment.

  1. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells

    Science.gov (United States)

    Cheng, Tzuling; Sudderth, Jessica; Yang, Chendong; Mullen, Andrew R.; Jin, Eunsook S.; Matés, José M.; DeBerardinis, Ralph J.

    2011-01-01

    Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how “glutamine-addicted” cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis. PMID:21555572

  2. Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

    Science.gov (United States)

    Lazar, Nathaniel; Fay, Allison; Nandakumar, Madhumitha; Boyle, Kerry E; Xavier, Joao; Rhee, Kyu; Glickman, Michael S

    2017-12-01

    Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. © 2017 John Wiley & Sons Ltd.

  3. Efficient acetylation of primary amines and amino acids in ...

    Indian Academy of Sciences (India)

    bDepartment of Clinical and Experimental Pharmacology, School of Tropical Medicine ... As a result ... methods of acetylation of amines are known using ace- ... vents we report here, environmentally benign, eco- ... It was filtered under suction,.

  4. Purification and properties of an O-acetyl-transferase from Escherichia coli that can O-acetylate polysialic acid sequences

    International Nuclear Information System (INIS)

    Higa, H.; Varki, A.

    1986-01-01

    Certain strains of bacteria synthesize an outer polysialic acid (K1) capsule. Some strains of K1 + E.coli are also capable of adding O-acetyl-esters to the exocyclic hydroxyl groups of the sialic acid residues. Both the capsule and the O-acetyl modification have been correlated with differences in antigenicity and pathogenicity. The authors have developed an assay for an O-acetyl-transferase in E.coli that transfers O-[ 3 H]acetyl groups from [ 3 H]acetyl-Coenzyme A to colominic acid (fragments of the polysialic acid capsule). Using this assay, the enzyme was solubilized, and purified ∼ 600-fold using a single affinity chromatography step with Procion Red-A Agarose. The enzyme also binds to Coenzyme A Sepharose, and can be eluted with high salt or Coenzyme A. The partially purified enzyme has a pH optimum of 7.0 - 7.5, is unaffected by divalent cations, is inhibited by high salt concentrations, is inhibited by Coenzyme A (50% inhibition at 100 μM), and shows an apparent Km for colominic acid of 3.7 mM (sialic acid concentration). This enzyme could be involved in the O-acetyl +/- form variation seen in some strains of K1 + E.coli

  5. Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

    Directory of Open Access Journals (Sweden)

    Marek M Galka

    Full Text Available Abscisic acid ((+-ABA is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC, x-ray crystallography and in silico modelling to identify putative (+-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP substrate. Functionally, (+-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM, but more potent inhibition of Rubisco activation (Ki of ~ 130 μM. Comparative structural analysis of Rubisco in the presence of (+-ABA with RuBP in the active site revealed only a putative low occupancy (+-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+-ABA binding site in the RuBP binding pocket. Overall we conclude that (+-ABA interacts with Rubisco. While the low occupancy (+-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

  6. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    Science.gov (United States)

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Physical exercise reduces pyruvate carboxylase (PCB) and contributes to hyperglycemia reduction in obese mice.

    Science.gov (United States)

    Muñoz, Vitor Rosetto; Gaspar, Rafael Calais; Crisol, Barbara Moreira; Formigari, Guilherme Pedron; Sant'Ana, Marcella Ramos; Botezelli, José Diego; Gaspar, Rodrigo Stellzer; da Silva, Adelino S R; Cintra, Dennys Esper; de Moura, Leandro Pereira; Ropelle, Eduardo Rochete; Pauli, José Rodrigo

    2018-07-01

    The present study evaluated the effects of exercise training on pyruvate carboxylase protein (PCB) levels in hepatic tissue and glucose homeostasis control in obese mice. Swiss mice were distributed into three groups: control mice (CTL), fed a standard rodent chow; diet-induced obesity (DIO), fed an obesity-inducing diet; and a third group, which also received an obesity-inducing diet, but was subjected to an exercise training protocol (DIO + EXE). Protocol training was carried out for 1 h/d, 5 d/wk, for 8 weeks, performed at an intensity of 60% of exhaustion velocity. An insulin tolerance test (ITT) was performed in the last experimental week. Twenty-four hours after the last physical exercise session, the animals were euthanized and the liver was harvested for molecular analysis. Firstly, DIO mice showed increased epididymal fat and serum glucose and these results were accompanied by increased PCB and decreased p-Akt in hepatic tissue. On the other hand, physical exercise was able to increase the performance of the mice and attenuate PCB levels and hyperglycemia in DIO + EXE mice. The above findings show that physical exercise seems to be able to regulate hyperglycemia in obese mice, suggesting the participation of PCB, which was enhanced in the obese condition and attenuated after a treadmill running protocol. This is the first study to be aimed at the role of exercise training in hepatic PCB levels, which may be a novel mechanism that can collaborate to reduce the development of hyperglycemia and type 2 diabetes in DIO mice.

  8. Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

    Directory of Open Access Journals (Sweden)

    Ansaya Thonpho

    Full Text Available Pyruvate carboxylase (PC is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an activator element(s for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y, as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription.

  9. Evidence for allosterism in ribulose-1,5-bisphosphate carboxylase/oxygenase from comfrey

    International Nuclear Information System (INIS)

    Mueller, D.D.; Bolden, T.D.

    1986-01-01

    Evidence has been obtained suggesting that ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an allosteric enzyme in the sense that it shows cooperative active site binding, cooperative interactions between the activation and active sites and significant binding of some metabolites at a second site. Investigation of the binding of a potent competitive inhibitor. 2-carboxymannitol-1,6-bisphosphate (CMBP) by 31 P-NMR indicated essentially 1:1 binding with the active sites of comfrey RuBisCo. Among the interactions of competitive inhibitors, as measured by difference UV spectroscopy, the binding curves for ortho-phosphate and ribose-5-phosphate were better fitted by a Monod-Wyman-Changeux model than by an independent site model, whereas the binding of CMBP and 2-phosphoglycolate were not. Difference UV methods also were used to study activation by CO 2 which at pH 7.9 in 10 mM MgCl 2 showed positive cooperativity with k = 100 +/- 3 μM (based on pK/sub a/ = 6.4 for the CO 2 -HCO 3 - equilibrium) and L = 3.5 +/- 0.7. Addition of saturating amounts of CMBP and lowering the MgCl 2 to 2 mM still gave a sigmoidal curve but it was shifted to higher CO 2 concentrations (k = 124 +/- 2 μM and L = 31 +/- 3). In the absence of CMBP the same conditions gave k = 26 +/- 2 μM for L = 3.5. Conversely, k was 0.96 +/- 0.08 μM for CMBP in 0.5 mM MgCl 2 without added NaHCO 3 but was 21 +/- 0.06 μM in 10 MgCl 2 and 2 mM NaHCO 3 , pH 7.3

  10. Effect of temperature and pH on the actiity of ribulose 1,5-diphosphate carboxylase from the thermophilic hydrogen bacterium Pseudomonas thermophila

    Energy Technology Data Exchange (ETDEWEB)

    Romanova, A K; Emnova, E E; Zykalova, K A

    1980-01-01

    The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completey loses its activity at 80/sup 0/C. The activity of RDP carboxylase at 60/sup 0/C increases by 40% during the first 10 min of heating in the presence of Mg/sup 2 +/ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50 to 55/sup 0/C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 keal.mole/sup -1/, but 11.6 kcal.mole/sup -1/ in frozen preparations. The optimal pH is 7.0 to 7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps, thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temeprature (the enzyme activity is negligible at 30/sup 0/C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.

  11. A Turkish Patient With Succinyl-CoA:3-Oxoacid CoA Transferase Deficiency Mimicking Diabetic Ketoacidosis

    Directory of Open Access Journals (Sweden)

    Sahin Erdol MD

    2016-05-01

    Full Text Available Succinyl-CoA:3-oxoacid CoA transferase (SCOT deficiency is an autosomal recessive disorder of ketone body utilization that is clinically characterized with intermittent ketoacidosis crises. We report here the second Turkish case with SCOT deficiency. She experienced 3 ketoacidotic episodes: The first ketoacidotic crisis mimicked diabetic ketoacidosis because of the associated hyperglycemia. Among patients with SCOT deficiency, the blood glucose levels at the first crises were variable, and this case had the highest ever reported blood glucose level. She is a compound heterozygote with 2 novel mutations, c.517A>G (K173E and c.1543A>G (M515V, in exons 5 and 17 of the OXCT1 gene, respectively. In patient’s fibroblasts, SCOT activity was deficient and, by immunoblot analysis, SCOT protein was much reduced. The patient attained normal development and had no permanent ketosis. The accurate diagnosis of SCOT deficiency in this case had a vital impact on the management strategy and outcome.

  12. Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

    Directory of Open Access Journals (Sweden)

    Ewing Andrew G

    2008-12-01

    Full Text Available Abstract Background Primordial germ cells (PGCs are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

  13. Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie [Nankai; (Chinese Aca. Sci.)

    2012-10-15

    Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.

  14. Determination of the barrier height for acetyl radical dissociation from acetyl chloride photodissociation at 235 nm using velocity map imaging.

    Science.gov (United States)

    Tang, Xiaonan; Ratliff, Britni J; FitzPatrick, Benjamin L; Butler, Laurie J

    2008-12-18

    This work uses velocity map imaging to determine the barrier height for acetyl radical, CH3CO, dissociation to CH3 + CO. Photodissociation of acetyl chloride at 235 nm generates acetyl radicals with an internal energy distribution spanning this barrier. We determine the velocity and internal energy distribution of all nascent acetyl radicals, stable and unstable, by measuring the velocities of the Cl(2P3/2) and Cl(2P1/2) cofragments. These Cl cofragments are detected with 2 + 1 resonance-enhanced multiphoton ionization (REMPI) in a spin-orbit branching ratio Cl(2P3/2):Cl(2P1/2) of 3.3 +/- 0.2. Using 157 nm photoionization, we then detect the recoil velocities of the energetically stable acetyl radicals. The radicals and momentum matched Cl atoms evidence parallel angular distributions. Comparison of the total recoil translational energy distribution P(E(T)) for all radicals to that obtained from the detection of stable radicals yields an onset for dissociation at a translational energy of 25.0 +/- 0.4 kcal/mol. From this onset we can calculate the barrier height for CH3CO --> CH3 + CO, but this relies on prior determinations of the C-Cl bond energy of acetyl chloride. Using an experimental bond dissociation energy of 83.4 +/- 0.2 kcal/mol yields a dissociation barrier of 14.2 +/- 0.5 kcal/mol. Our data evidence that a portion of the acetyl radicals formed with total internal energy above the barrier are stable due to the partitioning of energy into rotation during the C-Cl bond fission of the precursor. Thus, the internal energy onset for dissociation is not as sharp as was assumed in prior determinations of the barrier. The experimentally determined onset is compared with that predicted from electronic structure calculations at the G3//B3LYP and CCSD(T) levels of theory.

  15. Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation

    International Nuclear Information System (INIS)

    Waterborg, J.H.; Robertson, A.J.; Tatar, D.L.; Borza, C.M.; Davie, J.R.

    1995-01-01

    Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a since threonine as residue 28 instead of the serine(28)-alanine(29) sequence, present in all other known plant and animal H3 histones

  16. Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

    CERN Document Server

    Foulon, V; Croes, K; Waelkens, E

    1999-01-01

    Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

  17. Mechanism of action of clostridial glycine reductase: Isolation and characterization of a covalent acetyl enzyme intermediate

    International Nuclear Information System (INIS)

    Arkowitz, R.A.; Abeles, R.H.

    1991-01-01

    Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + P i + 2e - → acetyl phosphate + NH 4 + . Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. The authors now demonstrate that protein C catalyzes exchange of [ 32 P]P i into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, they have isolated acetyl protein C and shown that it is qualitatively, catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with P i to give acetyl phosphate. When [ 14 C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. Treatment with KBH 4 removes all the radioactivity associated with protein C, resulting in the formation of [ 14 C]ethanol. They conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [ 3 H]H 2 O into acetyl phosphate. This exchange reaction supports the proposal that an enol of the acetyl enzyme is an intermediate in the reaction sequence

  18. [Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

    Science.gov (United States)

    Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

    2003-02-01

    Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

  19. SIRT3 deacetylates mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 and regulates ketone body production

    DEFF Research Database (Denmark)

    Shimazu, Tadahiro; Hirschey, Matthew D; Hua, Lan

    2010-01-01

    with SIRT3 and in vivo by overexpression of SIRT3. Deacetylation of HMGCS2 lysines 310, 447, and 473 by incubation with wild-type SIRT3 or by mutation to arginine enhances its enzymatic activity. Molecular dynamics simulations show that in silico deacetylation of these three lysines causes conformational...... changes of HMGCS2 near the active site. Mice lacking SIRT3 show decreased β-hydroxybutyrate levels during fasting. Our findings show SIRT3 regulates ketone body production during fasting and provide molecular insight into how protein acetylation can regulate enzymatic activity....

  20. Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure, regulation and genetic engineering.

    Science.gov (United States)

    Rajagopalan, A V; Devi, M T; Raghavendra, A S

    1994-02-01

    Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C4-specific, C3 or etiolated, CAM and root forms. C4 leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C4-dicot Flaveria trinervia. Selective expression of ppc in only C4-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C4-PEPC pinpoint the phosphorylatable serine near the N-terminus, C4-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO2, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO3 (-)-dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C4 ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C4 PEPC and the transgenic tobacco plants expressed both C3 and C4 isoforms. Site-directed mutagenesis of ppc indicates the importance of His(138), His(579) and Arg(587) in catalysis and/or substrate-binding by the E. coli enzyme, Ser(8) in the regulation of sorghum PEPC. Important areas for further research on C4 PEPC are: mechanism of transduction of light signal during

  1. Evidence for allosterism in ribulose-1,5-bisphosphate carboxylase/oxygenase from comfrey

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, D.D.; Bolden, T.D.

    1986-05-01

    Evidence has been obtained suggesting that ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an allosteric enzyme in the sense that it shows cooperative active site binding, cooperative interactions between the activation and active sites and significant binding of some metabolites at a second site. Investigation of the binding of a potent competitive inhibitor. 2-carboxymannitol-1,6-bisphosphate (CMBP) by /sup 31/P-NMR indicated essentially 1:1 binding with the active sites of comfrey RuBisCo. Among the interactions of competitive inhibitors, as measured by difference UV spectroscopy, the binding curves for ortho-phosphate and ribose-5-phosphate were better fitted by a Monod-Wyman-Changeux model than by an independent site model, whereas the binding of CMBP and 2-phosphoglycolate were not. Difference UV methods also were used to study activation by CO/sub 2/ which at pH 7.9 in 10 mM MgCl/sub 2/ showed positive cooperativity with k = 100 +/- 3 ..mu..M (based on pK/sub a/ = 6.4 for the CO/sub 2/-HCO/sub 3//sup -/ equilibrium) and L = 3.5 +/- 0.7. Addition of saturating amounts of CMBP and lowering the MgCl/sub 2/ to 2 mM still gave a sigmoidal curve but it was shifted to higher CO/sub 2/ concentrations (k = 124 +/- 2 ..mu..M and L = 31 +/- 3). In the absence of CMBP the same conditions gave k = 26 +/- 2 ..mu..M for L = 3.5. Conversely, k was 0.96 +/- 0.08 ..mu..M for CMBP in 0.5 mM MgCl/sub 2/ without added NaHCO/sub 3/ but was 21 +/- 0.06 ..mu..M in 10 MgCl/sub 2/ and 2 mM NaHCO/sub 3/, pH 7.3.

  2. Small molecule inhibitors of bromodomain-acetyl-lysine interactions.

    Science.gov (United States)

    Brand, Michael; Measures, Angelina R; Measures, Angelina M; Wilson, Brian G; Cortopassi, Wilian A; Alexander, Rikki; Höss, Matthias; Hewings, David S; Rooney, Timothy P C; Paton, Robert S; Conway, Stuart J

    2015-01-16

    Bromodomains are protein modules that bind to acetylated lysine residues. Their interaction with histone proteins suggests that they function as "readers" of histone lysine acetylation, a component of the proposed "histone code". Bromodomain-containing proteins are often found as components of larger protein complexes with roles in fundamental cellular process including transcription. The publication of two potent ligands for the BET bromodomains in 2010 demonstrated that small molecules can inhibit the bromodomain-acetyl-lysine protein-protein interaction. These molecules display strong phenotypic effects in a number of cell lines and affect a range of cancers in vivo. This work stimulated intense interest in developing further ligands for the BET bromodomains and the design of ligands for non-BET bromodomains. Here we review the recent progress in the field with particular attention paid to ligand design, the assays employed in early ligand discovery, and the use of computational approaches to inform ligand design.

  3. Synthesis of polyrotaxanes from acetyl-β-cyclodextrin

    Science.gov (United States)

    Ristić, I. S.; Nikolić, L.; Nikolić, V.; Ilić, D.; Budinski-Simendić, J.

    2011-12-01

    Polyrotaxanes are intermediary products in the synthesis of topological gels. They are created by inclusion complex formation of hydrophobic linear macromolecules with cyclodextrins or their derivatives. Then, pairs of cyclodextrin molecules with covalently linkage were practically forming the nodes of the semi-flexible polymer network. Such gels are called topological gels and they can absorb huge quantities of water due to the net flexibility allowing the poly(ethylene oxide) chains to slide through the cyclodextrin cavities, without being pulled out altogether. For polyrotaxane formation poly(ethylene oxide) was used like linear macromolecules. There are hydroxyl groups at poly(ethylene oxide) chains, whereby the linking of the voluminous molecules should be made. To avoid the reaction of cyclodextrin OH groups with stoppers, they should be protected by, e.g., acetylation. In this work, the acetylation of the OH groups of β-cyclodextrin was performed by acetic acid anhydride with iodine as the catalyst. The acetylation reaction was assessed by the FTIR and HPLC method. By the HPLC analysis was found that the acetylation was completed in 20 minutes. Inserting of poly(ethylene oxide) with 4000 g/mol molecule mass into acetyl-β-cyclodextrin with 2:1 poly(ethylene oxide) monomer unit to acetyl-β-cyclodextrin ratio was also monitored by FTIR, and it was found that the process was completed in 12 h at the temperature of 10°C. If the process is performed at temperatures above 10°C, or for periods longer than 12 hours, the process of uncontrolled hydrolysis of acetate groups was initiated.

  4. Effect of biotin on activity and gene expression of biotin-dependent carboxylases in the liver of dairy cows.

    Science.gov (United States)

    Ferreira, G; Weiss, W P

    2007-03-01

    Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40 +/- 2 kg/d of milk yield and 162 +/- 35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocarboxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

  5. Malonyl CoA decarboxylase deficiency: C to T transition in intron 2 of the MCD gene.

    Science.gov (United States)

    Surendran, S; Sacksteder, K A; Gould, S J; Coldwell, J G; Rady, P L; Tyring, S K; Matalon, R

    2001-09-15

    Malonyl CoA decarboxylase (MCD) is an enzyme involved in the metabolism of fatty acids synthesis. Based on reports of MCD deficiency, this enzyme is particular important in muscle and brain metabolism. Mutations in the MCD gene result in a deficiency of MCD activity, that lead to psychomotor retardation, cardiomyopathy and neonatal death. To date however, only a few patients have been reported with defects in MCD. We report here studies of a patient with MCD deficiency, who presented with hypotonia, cardiomyopathy and psychomotor retardation. DNA sequencing of MCD revealed a homozygous intronic mutation, specifically a -5 C to T transition near the acceptor site for exon 3. RT-PCR amplification of exons 2 and 3 revealed that although mRNA from a normal control sample yielded one major DNA band, the mutant mRNA sample resulted in two distinct DNA fragments. Sequencing of the patient's two RT-PCR products revealed that the larger molecular weight fragments contained exons 2 and 3 as well as the intervening intronic sequence. The smaller size band from the patient contained the properly spliced exons, similar to the normal control. Western blotting analysis of the expressed protein showed only a faint band in the patient sample in contrast to a robust band in the control. In addition, the enzyme activity of the mutant protein was lower than that of the control protein. The data indicate that homozygous mutation in intron 2 disrupt normal splicing of the gene, leading to lower expression of the MCD protein and MCD deficiency. Copyright 2001 Wiley-Liss, Inc.

  6. Predicting post-translational lysine acetylation using support vector machines

    DEFF Research Database (Denmark)

    Gnad, Florian; Ren, Shubin; Choudhary, Chunaram

    2010-01-01

    spectrometry to identify 3600 lysine acetylation sites on 1750 human proteins covering most of the previously annotated sites and providing the most comprehensive acetylome so far. This dataset should provide an excellent source to train support vector machines (SVMs) allowing the high accuracy in silico...

  7. High regioselective acetylation of vitamin A precursors using lipase ...

    African Journals Online (AJOL)

    Administrator

    2011-09-26

    Sep 26, 2011 ... High regioselective acetylation of vitamin A precursors using lipase B from Candida antarctica in organic media. Jingpeng Sun, Keju Jing* and Yinghua Lu. Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen. University, Xiamen 361005, P. R. ...

  8. Surface effects in the acetylation of granular potato starch

    NARCIS (Netherlands)

    Steeneken, P.A.M.; Woortman, A.J.J.

    2008-01-01

    The occurrence of surface effects in the acetylation of granular potato starch with acetic anhydride to degrees of substitution 0.04-0.2 was studied by two different approaches. The first approach involved the fractionation of granular starch acetates into five different size classes and analysis of

  9. Effect Of Nicotine And Tobacco Consumption On Brain Acetyl ...

    African Journals Online (AJOL)

    The effect of nicotine and tobacco consumption on brain acetyl cholinesterase and serum alkaline phosphatase in rats was studied. Rats were divided into three groups and the first group was fed rat chow and water ad libitum and an oral administration of 2ml of 0.1%(v/v) nicotine per 100g body weight of rats per day.

  10. Genetic control of differential acetylation in diabetic rats.

    Directory of Open Access Journals (Sweden)

    Pamela J Kaisaki

    Full Text Available Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.

  11. Effect of acetyl groups of wood on furfural preparation

    Energy Technology Data Exchange (ETDEWEB)

    Skvortsov, S.V.; Kolchina, N.P.; Miroshnichenko, V.G.

    1981-01-01

    The deacetylation (4% Na/sub 2/CO/sub 3/, 60 degrees) of birch sawdust prior to hydrolysis decreased the yield of furfural, presumably due to thermal degradation of pentosans and buildup of HCOOH in the wood stock. Thus, while untreated birch sawdust gave 6.6% furfural, the acetylated sawdust gave only 4.5% furfural.

  12. Effect of acetylation and varietal differences on the pasting ...

    African Journals Online (AJOL)

    The pasting properties of starch from eight varieties of corn; Okomasa, Obatanpa, Dodzi, Mamaba, Dadaba, Dorke, Golden crystal, and CIDA-ba were studied to establish the effects of acetylation and varietal differences on the pasting properties. Native starches extracted from the corn varieties were modified with 10% v/v ...

  13. Acetylation of wood components and fourier transform infra-red ...

    African Journals Online (AJOL)

    In this study, the reactivity of wood components with acetic anhydride or vinyl acetate was studied. It was found that the reactivity of wood components was virgin wood flour > holocellulose >> a-cellulose. Acetylation of Turkish pine or cedar wood flour with acetic anhydride was significantly improved in the presence of ...

  14. DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity▿ †

    OpenAIRE

    Sun, Yingli; Xu, Ye; Roy, Kanaklata; Price, Brendan D.

    2007-01-01

    The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-ly...

  15. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins

    Directory of Open Access Journals (Sweden)

    Michael N. Davies

    2016-01-01

    Full Text Available Lysine acetylation (AcK, a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT, an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

  16. Comprehensive Proteomic Analysis of Lysine Acetylation in the Foodborne Pathogen Trichinella spiralis

    Directory of Open Access Journals (Sweden)

    Yong Yang

    2018-01-01

    Full Text Available Lysine acetylation is a dynamic and highly conserved post-translational modification that plays a critical role in regulating diverse cellular processes. Trichinella spiralis is a foodborne parasite with a considerable socio-economic impact. However, to date, little is known regarding the role of lysine acetylation in this parasitic nematode. In this study, we utilized a proteomic approach involving anti-acetyl lysine-based enrichment and highly sensitive mass spectrometry to identify the global acetylated proteome and investigate lysine acetylation in T. spiralis. In total, 3872 lysine modification sites were identified in 1592 proteins that are involved in a wide variety of biological processes. Consistent with the results of previous studies, a large number of the acetylated proteins appear to be involved in metabolic and biosynthetic processes. Interestingly, according to the functional enrichment analysis, 29 acetylated proteins were associated with phagocytosis, suggesting an important role of lysine acetylation in this process. Among the identified proteins, 15 putative acetylation motifs were detected. The presence of serine downstream of the lysine acetylation site was commonly observed in the regions surrounding the sites. Moreover, protein interaction network analysis revealed that various interactions are regulated by protein acetylation. These data represent the first report of the acetylome of T. spiralis and provide an important resource for further explorations of the role of lysine acetylation in this foodborne pathogen.

  17. Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves

    NARCIS (Netherlands)

    Tsuprun, V.L.; Boekema, E.J.; Samsonidze, T.G.; Pushkin, A.V.

    1991-01-01

    The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of

  18. The MDM2-p53-pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    DEFF Research Database (Denmark)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao

    2016-01-01

    deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates...

  19. 3-Methylcrotonyl-coenzyme A carboxylase deficiency in Amish/Mennonite adults identified by detection of increased acylcarnitines in blood spots of their children.

    Science.gov (United States)

    Gibson, K M; Bennett, M J; Naylor, E W; Morton, D H

    1998-03-01

    Isolated 3-methylcrotonyl coenzyme A carboxylase (MCC) deficiency was documented in four adult women from the Amish/Mennonite population of Lancaster County, Pennsylvania. Metabolic and enzymatic investigations in these individuals were instituted after the detection of abnormal acylcarnitine profiles in blood spots obtained from their newborn children, in whom MCC activity was normal.

  20. Daily rhythm of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants : Immunological evidence for the absence of a rhythm in protein synthesis.

    Science.gov (United States)

    Brulfert, J; Vidal, J; Gadal, P; Queiroz, O

    1982-11-01

    Immunotitration of phosphoenolpyruvate carboxylase (EC 4.1.1.31) extracted from leaves of Kalanchoe blossfeldiana v. Poelln. cv. Tom Thumb. It was established that at different times of the day-night cycle the daily rhythm of enzyme capacity does not result from a rhythm in protein synthesis, but rather from changes in the specific activity of the enzyme.

  1. Sequential Dy(OTf)3 -Catalyzed Solvent-Free Per-O-Acetylation and Regioselective Anomeric De-O-Acetylation of Carbohydrates.

    Science.gov (United States)

    Yan, Yi-Ling; Guo, Jiun-Rung; Liang, Chien-Fu

    2017-09-19

    Dysprosium(III) trifluoromethanesulfonate-catalyzed per-O-acetylation and regioselective anomeric de-O-acetylation of carbohydrates can be tuned by adjusting the reaction medium. In this study, the per-O-acetylation of unprotected sugars by using a near-stoichiometric amount of acetic anhydride under solvent-free conditions resulted in the exclusive formation of acetylated saccharides as anomeric mixtures, whereas anomeric de-O-acetylation in methanol resulted in a moderate-to-excellent yield. Reactions with various unprotected monosaccharides or disaccharides followed by a semi-one-pot sequential conversion into the corresponding acetylated glycosyl hemiacetal also resulted in high yields. Furthermore, the obtained hemiacetals could be successfully transformed into trichloroimidates after Dy(OTf) 3 -catalyzed glycosylation. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Studies investigating the excretion of acetyl urea in the milk of dairy cows receiving oral doses of 14C acetyl urea

    International Nuclear Information System (INIS)

    Bergner, H.; Kijora, C.; Goersch, R.

    1976-01-01

    2 experimental cows were fed acetyl urea several weeks before the trial was started. The first cow received a daily amount of 200 g and the second cow 855 g. On the first day of experiment both cows were given 5 mCi of 14 C acetyl urea intraruminally. Up to 6 hrs after the beginning of the experiment acetyl urea in blood plasma was shown to contain a higher proportion of 14 C activity than urea. 0.21 g urea and 0.18 g acetyl urea were contained in 1 kg of milk from cow No 1 while 1 kg of milk from cow No 2 contained 0.18 g urea and 0.12 g acetyl urea. The feeding of acetyl urea to dairy cows is not recommended on the basis of the fact that any further contamination of human nutrition with foreign substances should be possibly avoided. (author)

  3. Studies investigating the excretion of acetyl urea in the milk of dairy cows receiving oral doses of /sup 14/C acetyl urea

    Energy Technology Data Exchange (ETDEWEB)

    Bergner, H; Kijora, C; Goersch, R [Humboldt-Universitaet, Berlin (German Democratic Republic). Sektion Tierproduktion und Veterinaermedizin

    1976-01-01

    2 experimental cows were fed acetyl urea several weeks before the trial was started. The first cow received a daily amount of 200 g and the second cow 855 g. On the first day of experiment both cows were given 5 mCi of /sup 14/C acetyl urea intraruminally. Up to 6 hrs after the beginning of the experiment acetyl urea in blood plasma was shown to contain a higher proportion of /sup 14/C activity than urea. 0.21 g urea and 0.18 g acetyl urea were contained in 1 kg of milk from cow No 1 while 1 kg of milk from cow No 2 contained 0.18 g urea and 0.12 g acetyl urea. The feeding of acetyl urea to dairy cows is not recommended on the basis of the fact that any further contamination of human nutrition with foreign substances should be possibly avoided.

  4. Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase.

    Science.gov (United States)

    Thornburg, Chelsea K; Walter, Tyler; Walker, Kevin D

    2017-11-07

    In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.

  5. RNAi inhibition of feruloyl CoA 6'-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots.

    Science.gov (United States)

    Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R

    2017-05-01

    Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.

  6. 2-Acetylthiamin pyrophosphate (acetyl-TPP) pH-rate profile for hydrolysis of acetyl-TPP and isolation of acetyl-TPP as a transient species in pyruvate dehydrogenase catalyzed reactions

    International Nuclear Information System (INIS)

    Gruys, K.J.; Datta, A.; Frey, P.A.

    1989-01-01

    Rate constants for the hydrolysis of acetyl-TPP were measured pH values of 2.5 and 7.5 and plotted as log k obs versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pK a of 4.73 within acetyl-TPP that is remote from the acetyl group, the aminopyrimidine ring, which is promoted below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP have facilitated the isolation and identification of [1- 14 C]acetyl-TPP from acid-quenched enymatic reaction mixtures at steady states. [1- 14 C]Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E 1 ). The pH-rate profile for hydrolysis of [1- 14 C]-acetyl-TPP, isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the 14 C-labeled enzymic species

  7. Acetyl coenzyme A synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single Gcn5-type N-acetyltransferase (GNAT) domain in Saccharopolyspora erythraea.

    Science.gov (United States)

    You, Di; Yao, Li-Li; Huang, Dan; Escalante-Semerena, Jorge C; Ye, Bang-Ce

    2014-09-01

    Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD(+)-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys(237), Lys(380), Lys(611), and Lys(628) was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Differential transcription and message stability of two genes encoding soybean ribulose 1,5-bisphosphate carboxylase small subunit

    International Nuclear Information System (INIS)

    Shirley, B.W.; Berry-Lowe, S.L.; Grandbastien, M.A.; Zurfluh, L.L.; Shah, D.M.; Meagher, R.B.

    1987-01-01

    The expression of two closely related soybean ribulose bisphosphate carboxylase small subunit (Rubisco ss) genes, SRS1 and SRS4, has been compared. These genes account for approximately 2-4% of the total transcription in light grown leaves, SRS4 being twice as transcriptionally active as SRS1. The transcription of these genes is reduced more than 30 fold after a pulse of far-red light or extended periods of darkness. When etiolated seedlings are shifted to the light the transcription of both genes increases 30-50 fold. Despite this 30-fold range in transcriptional expression the steady state mRNA levels in light and dark grown tissue differ by less than 8 fold. This suggests that the mRNAs are less stable in light grown tissue. 38 refs., 5 figs

  9. Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia: Mollusca).

    Science.gov (United States)

    Cavanaugh, C M; Abbott, M S; Veenhuis, M

    1988-10-01

    The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP(2)Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large subunit of RbuP(2)Case from tobacco (Nicotiana tabacum) cross-reacted on immunoblots with a protein of similar molecular mass occurring in extracts of the symbiont-containing gill tissue of S. velum. No cross-reactivity was detected in symbiont-free tissue extracts. The antiserum also cross-reacted in immunoblots with proteins of Thiobacillus neapolitanus, a free-living sulfuroxidizing chemoautotroph whose RbuP(2)Case has been well characterized. In protein A-gold immunoelectron microscopy studies, this antiserum consistently labeled the symbionts but not surrounding host gill tissue, indicating that the symbionts are responsible for the RbuP(2)Case activity.

  10. Phosphorylation-dephosphorylation process as a probable mechanism for the diurnal regulatory changes of phosphoenolpyruvate carboxylase in CAM plants.

    Science.gov (United States)

    Brulfert, J; Vidal, J; Le Marechal, P; Gadal, P; Queiroz, O; Kluge, M; Kruger, I

    1986-04-14

    Day and night forms of phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEPC) were extracted from leaves of the CAM plants Kalanchoe daigremontiana, K. tubiflora and K. blossfeldiana previously fed with [32P] labelled phosphate solution. A one-step immunochemical purification followed by SDS polyacrylamide gel electrophoresis and autoradiography showed that, in all species, the night form of the enzyme was phosphorylated and not the day form. Limited acid hydrolysis of the night form and two-dimensional separation identified predominantly labelled phosphoserine and phosphothreonine. In vitro addition of exogenous acid phosphatase (EC 3.1.3.2) to desalted night form-containing extracts resulted within 30 min in a shift in PEPC enzymic properties similar to the in vivo changes from night to day form. It is suggested that phosphorylation-dephosphorylation of the enzyme could be the primary in vivo process which might explain the observed rhythmicity of enzymic properties.

  11. Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties.

    Science.gov (United States)

    Orfila, Caroline; Dal Degan, Florence; Jørgensen, Bodil; Scheller, Henrik Vibe; Ray, Peter M; Ulvskov, Peter

    2012-07-01

    A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.

  12. Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species.

    Science.gov (United States)

    Ueno, Osamu; Ishimaru, Ken

    2002-01-01

    The leafless amphibious sedge Eleocharis vivipara develops culms with C(4) traits and Kranz anatomy under terrestrial conditions, but develops culms with C(3) traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C(4) enzyme activities, while those of the submerged form have decreased C(4) enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C(3) pathway in the submerged form. To verify the presence of the C(3) cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C(4) control), but by only 20% in potato (C(3) control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C(4) and C(3) plants, at 58-81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO(2) by Rubisco and probably the C(3) cycle in the MC. These features are reminiscent of those in C(4)-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C(3) activity in the MC.

  13. Lipids Reprogram Metabolism to Become a Major Carbon Source for Histone Acetylation

    DEFF Research Database (Denmark)

    McDonnell, Eoin; Crown, Scott B; Fox, Douglas B

    2016-01-01

    Cells integrate nutrient sensing and metabolism to coordinate proper cellular responses to a particular nutrient source. For example, glucose drives a gene expression program characterized by activating genes involved in its metabolism, in part by increasing glucose-derived histone acetylation....... Here, we find that lipid-derived acetyl-CoA is a major source of carbon for histone acetylation. Using (13)C-carbon tracing combined with acetyl-proteomics, we show that up to 90% of acetylation on certain histone lysines can be derived from fatty acid carbon, even in the presence of excess glucose...

  14. Acetylation Increases EWS-FLI1 DNA Binding and Transcriptional Activity

    International Nuclear Information System (INIS)

    Schlottmann, Silke; Erkizan, Hayriye V.; Barber-Rotenberg, Julie S.; Knights, Chad; Cheema, Amrita; Üren, Aykut; Avantaggiati, Maria L.; Toretsky, Jeffrey A.

    2012-01-01

    Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  15. Acetylated tubulin is essential for touch sensation in mice.

    Science.gov (United States)

    Morley, Shane J; Qi, Yanmei; Iovino, Loredana; Andolfi, Laura; Guo, Da; Kalebic, Nereo; Castaldi, Laura; Tischer, Christian; Portulano, Carla; Bolasco, Giulia; Shirlekar, Kalyanee; Fusco, Claudia M; Asaro, Antonino; Fermani, Federica; Sundukova, Mayya; Matti, Ulf; Reymond, Luc; De Ninno, Adele; Businaro, Luca; Johnsson, Kai; Lazzarino, Marco; Ries, Jonas; Schwab, Yannick; Hu, Jing; Heppenstall, Paul A

    2016-12-13

    At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch.

  16. Acetyl-L-carnitine improves aged brain function.

    Science.gov (United States)

    Kobayashi, Satoru; Iwamoto, Machiko; Kon, Kazuo; Waki, Hatsue; Ando, Susumu; Tanaka, Yasukazu

    2010-07-01

    The effects of acetyl-L-carnitine (ALCAR), an acetyl derivative of L-carnitine, on memory and learning capacity and on brain synaptic functions of aged rats were examined. Male Fischer 344 rats were given ALCAR (100 mg/kg bodyweight) per os for 3 months and were subjected to the Hebb-Williams tasks and AKON-1 task to assess their learning capacity. Cholinergic activities were determined with synaptosomes isolated from brain cortices of the rats. Choline parameters, the high-affinity choline uptake, acetylcholine (ACh) synthesis and depolarization-evoked ACh release were all enhanced in the ALCAR group. An increment of depolarization-induced calcium ion influx into synaptosomes was also evident in rats given ALCAR. Electrophysiological studies using hippocampus slices indicated that the excitatory postsynaptic potential slope and population spike size were both increased in ALCAR-treated rats. These results indicate that ALCAR increases synaptic neurotransmission in the brain and consequently improves learning capacity in aging rats.

  17. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    DEFF Research Database (Denmark)

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

    2015-01-01

    Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acety......1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases....

  18. EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

    Science.gov (United States)

    Wang, Yi-Long; Chen, Hui; Zhan, Yi-Qun; Yin, Rong-Hua; Li, Chang-Yan; Ge, Chang-Hui; Yu, Miao; Yang, Xiao-Ming

    2016-08-17

    EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

  19. Chromatin decondensed by acetylation shows an elevated radiation response

    International Nuclear Information System (INIS)

    Nackerdien, Z.; Michie, J.; Boehm, L.

    1989-01-01

    V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair

  20. Autoradiographic study of nuclear protein acetylation during Locust spermiogenesis

    International Nuclear Information System (INIS)

    Bouvier, D.; Chevaillier, P.

    1975-01-01

    Autoradiographic studies, at the light and electron microscope level, demonstrate that spermatid nuclei of the Locust Locusta migratoria incorporate 3 H-acetate, especially during the first stages of spermiogenesis. The highest level of acetate incorporation is observed during stage II of spermiogenesis. During this stage and the following, the spermatid nucleus undergoes a number of structural and chemical modifications: chromatin decondenses and somatic histones are progressively replaced by newly synthesized arginine-rich proteins. Therefore, the higher degree of acetylation of nuclear components coincides with chromatin decondensation and precedes the protein transition occurring in later stages. Cytochemical and autoradiographic tests have been realized so as to localize 3 H-acetate in the nuclear components. Trichloracetic acid was used at various concentrations: the action of hydrochloric acid, pronase and DNase was also tested. The results support the idea that proteins, and among them histones, are the only nuclear components to be acetylated during spermiogenesis. Thus, histone acetylation seems to play an important role in modulating histone-DNA interactions and allowing histone replacement [fr

  1. Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri▿ †

    Science.gov (United States)

    Li, Fuli; Hinderberger, Julia; Seedorf, Henning; Zhang, Jin; Buckel, Wolfgang; Thauer, Rudolf K.

    2008-01-01

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0′ = −410 mV) with NADH (E0′ = −320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = −10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper. PMID:17993531

  2. Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri.

    Science.gov (United States)

    Li, Fuli; Hinderberger, Julia; Seedorf, Henning; Zhang, Jin; Buckel, Wolfgang; Thauer, Rudolf K

    2008-02-01

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.

  3. 1200 nt rat liver mRNA identified by differential hybridization exhibits coordinate regulation with 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase

    International Nuclear Information System (INIS)

    Tanaka, R.D.; Clarke, C.F.; Fogelman, A.M.; Edwards, P.A.

    1986-01-01

    Differential hybridization has been used to identify genes in rat liver that encode transcripts which are increased by the drugs cholestyramine and mevinolin and are decreased by dietary cholesterol. This approach should prove useful in isolating and identifying coordinately regulated genes involved in the isoprene biosynthetic pathway. Rat liver poly (A) + RNA was isolated from animals fed diets supplemented with either cholestyramine and mevinolin or with cholesterol. Radiolabeled cDNAs generated from these two RNA preparations were used to screen a rat cDNAs library. A preliminary screen of 10,000 recombinants has led to the identification of a clone with an insert of 1200 bp that hybridizes to a mRNA species of about 1200 nt. The level of this RNA species in rat liver is elevated by the drugs cholestyramine and mevinolin and is decreased by cholesterol feeding. This RNA species is also decreased by mevalonate administration to rats. The regulation of this 1200 nt mRNA species mirrors that of HMG CoA reductase and HMG CoA synthase. It seems very likely that this 1200 nt mRNA encodes a polypeptide which is involved in the isoprene biosynthetic pathway

  4. Correlation of changes in rate of sterol synthesis with changes in HMG CoA reductase activity in cultured lens epithelial cells

    International Nuclear Information System (INIS)

    Cenedella, R.J.; Hitchener, W.R.

    1986-01-01

    In the present study, the authors correlated changes in HMG CoA reductase activity with changes in relative rates of sterol synthesis measured from either 3 H 2 O or 1- 14 C-acetate for bovine lens epithelial cells cultured in the presence or absence of lipoproteins. Enzyme activity and rates of incorporation of 3 H 2 O or 1- 14 C-acetate into digitonin precipitable sterols were measured in cells on the 4th day of subculture in DMEM containing 9% whole calf serum (WM) or 9% lipoprotein deficient serum (LDM). In three experiments, HMG CoA reductase activity (U/10 6 cells) averaged 2.2 +/- 0.1 times greater for cells grown in LDM than WM. Sterol synthesis averaged 3.0 +/- 0.4 times greater when measured with 3 H 2 O and 4.0 +/- 1.1 times greater when measured with 14 C-acetate. Thus, 3 H 2 O and 14 C-acetate appear to be comparable substrates for estimating changes in relative rates of sterol synthesis by cultured cells. The larger increases in rates of sterol synthesis than in reductase activity in response to decreased cholesterol could reflect stimulation at additional metabolic steps in the cholesterol pathway beyond mevalonic acid

  5. Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation.

    Science.gov (United States)

    He, Hongzhen; Ding, Yi; Bartlam, Mark; Sun, Fei; Le, Yi; Qin, Xincheng; Tang, Hong; Zhang, Rongguang; Joachimiak, Andrzej; Liu, Jinyuan; Zhao, Nanming; Rao, Zihe

    2003-01-31

    Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

  6. Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for {beta}-lactam acetylation.

    Energy Technology Data Exchange (ETDEWEB)

    He, H.; Ding, Y.; Bartlam, M.; Sun, F.; Le, Y.; Qin, X.; Tang, H.; Zhang, R.; Joachimiak, A.; Liu, J.; Zhao, N.; Rao, Z.; Biosciences Division; Tsinghua Univ.; Chinese Academy of Science

    2003-01-31

    Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55 {angstrom} resolution. The binary complex forms a characteristic 'V' shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

  7. Density functional and ab initio study of the tautomeric forms of 3-acetyl tetronic and 3-acetyl tetramic acids

    International Nuclear Information System (INIS)

    Skylaris, Chris-Kriton; Igglessi-Markopoulou, Olga; Detsi, Anastasia; Markopoulos, John

    2003-01-01

    We propose all the accessible paths of interconversion between the tautomers of 3-acetyl tetronic and 3-acetyl tetramic acids by performing calculations with the density functional B3LYP method and the ab initio MP2 method. Our findings clarify at the atomic level the mechanisms of the equilibria between these tautomers, a topic so far only partially understood on the basis of studies by nuclear magnetic resonance (NMR) spectroscopy. We show that thermal effects via relative Gibbs free energies ΔG must be taken into account in order to reach good quantitative agreement with the available experimental information on the ratios of the most stable tautomers. The calculated 1 H and 13 C chemical shifts are in agreement with the experimental values from NMR spectroscopy

  8. Acetyl Groups in Typha capensis: Fate of Acetates during Organosolv and Ionosolv Pulping

    Directory of Open Access Journals (Sweden)

    Idi Guga Audu

    2018-06-01

    Full Text Available During biomass fractionation, any native acetylation of lignin and heteropolysaccharide may affect the process and the resulting lignin structure. In this study, Typha capensis (TC and its lignin isolated by milling (MWL, ionosolv (ILL and organosolv (EOL methods were investigated for acetyl group content using FT-Raman, 1H NMR, 2D-NMR, back-titration, and Zemplén transesterification analytical methods. The study revealed that TC is a highly acetylated grass; extractive free TC (TCextr and TC MWL exhibited similar values of acetyl content: 6 wt % and 8 wt % by Zemplén transesterification, respectively, and 11 wt % by back-titration. In contrast, lignin extracted from organosolv and [EMIm][OAc] pulping lost 80% of the original acetyl groups. With a high acetyl content in the natural state, TC could be an interesting raw material in biorefinery in which acetic acid could become an important by-product.

  9. Novel myelin penta- and hexa-acetyl-galactosyl-ceramides: structural characterization and immunoreactivity in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Podbielska, Maria; Dasgupta, Somsankar; Levery, Steven B

    2010-01-01

    Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous...... NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O...... Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked....

  10. Evaluation of gels obtained from acetylation of chitosan in heterogeneous medium

    International Nuclear Information System (INIS)

    Garcia, Rosangela Balaban; Silva, Dayse Luzia Pinheiro da; Costa, Marta

    2008-01-01

    Chitosan was acetylated during 2, 5 and 10 h and physical gels were obtained at different polymer concentrations in N,N-dimethylacetamide containing 5% of LiCl. Acetylation was confirmed by infrared spectroscopy and 13 C NMR, and degrees of acetylation in the range of 0.82-0.91 were determined by NMR. The O-acetylation degree (0.12-0.15) was exclusively determined by a volumetric method. Rheological studies showed that the storage modulus values were smaller for the more acetylated samples and increased with the temperature and the polymer concentration. All the gels presented storage modulus superior to loss modulus, evidencing more elastic than viscous characteristics. The results obtained in this work suggest a gelation process based on a balance between O and N-acetylation and intermolecular bonds. (author)

  11. Identification of the large subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase as a substrate for transglutaminase in Medicageo sativa L. (alfalfa)

    International Nuclear Information System (INIS)

    Margosiak, S.A.; Dharma, A.; Carver, M.R.B.; Gonzales, A.P.; Louie, D.; Kuehn, G.D.

    1990-01-01

    Extract prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [ 14 C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [ 14 C]putresciene incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts

  12. NEW EMBO MEMBER'S REVIEW: Acetylation: a regulatory modification to rival phosphorylation?

    OpenAIRE

    Kouzarides, Tony

    2000-01-01

    The fact that histones are modified by acetylation has been known for almost 30 years. The recent identification of enzymes that regulate histone acetylation has revealed a broader use of this modification than was suspected previously. Acetylases are now known to modify a variety of proteins, including transcription factors, nuclear import factors and α–tubulin. Acetylation regulates many diverse functions, including DNA recognition, protein–protein interaction and protein stability. There i...

  13. Autoimmune regulator is acetylated by transcription coactivator CBP/p300

    Energy Technology Data Exchange (ETDEWEB)

    Saare, Mario, E-mail: mario.saare@ut.ee [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); Rebane, Ana [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); SIAF, Swiss Institute of Allergy and Asthma Research, University of Zuerich, Davos (Switzerland); Rajashekar, Balaji; Vilo, Jaak [BIIT, Bioinformatics, Algorithmics and Data Mining group, Institute of Computer Science, University of Tartu, Tartu (Estonia); Peterson, Paert [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)

    2012-08-15

    The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE

  14. The dynamics of CO2 fixation in the Southern Ocean as indicated by carboxylase activities and organic carbon isotopic ratios

    International Nuclear Information System (INIS)

    Fontugne, M.

    1991-01-01

    Recent studies have suggested a direct relationship between the dissolved CO 2 concentration and carbon isotopic composition of phytoplankton in surface ocean. Thus, measurement of δ 13 C of planktonic organic matter in deep-sea ocean cores can potentially yield a record of the past atmospheric CO 2 variations. However, results are presented from 3 cruises in Indian and Atlantic sectors of the Southern Ocean (between 40-66degS) in which biochemical and physiological factors associated with photosynthetic processes lead to carbon isotopic fractionation by phytoplankton which cannot be directly related to variations within the mineral carbon pool. Simultaneous measurements of the carboxylase activities in the 13 C/ 12 C ratio of particulate organic carbon show that there is a large variability in phytoplankton carbon metabolism, especially on a seasonal scale, in spite of a relative uniformity of the environmental conditions. Phytoplankton carbon metabolism is clearly a main factor governing variations in the stable isotopic composition of organic matter in the euphotic layer. Interrelationships between light, Rubiso activity and δ 13 C are clearly shown by the data. Heterotrophic processes may also influence the carbon isotope mass balance, especially during the break-up of the ice pack. In addition to the influence of photosynthetic metabolism, the effect of the meridoneal temperature gradient is also verified by the data set. (author). 24 refs.; 5 figs

  15. Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism.

    Science.gov (United States)

    Ettema, Thijs J G; Makarova, Kira S; Jellema, Gera L; Gierman, Hinco J; Koonin, Eugene V; Huynen, Martijn A; de Vos, Willem M; van der Oost, John

    2004-11-01

    Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.

  16. Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

    Science.gov (United States)

    Qin, Na; Xu, Weigang; Hu, Lin; Li, Yan; Wang, Huiwei; Qi, Xueli; Fang, Yuhui; Hua, Xia

    2016-11-01

    Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

  17. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  18. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    International Nuclear Information System (INIS)

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H.

    1991-01-01

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC

  19. Preparation of radioactive acetyl-l-carnitine by an enzymatic exchange reaction

    International Nuclear Information System (INIS)

    Emaus, R.; Bieber, L.L.

    1982-01-01

    A rapid method for the preparation of [1- 14 C]acetyl-L-carnitine is described. The method involves exchange of [1- 14 C]acetic acid into a pool of unlabeled acetyl-L-carnitine using the enzymes acetyl-CoA synthetase and carnitine acetyltransferase. After isotopic equilibrium is attained, radioactive acetylcarnitine is separated from the other reaction components by chromatography on Dowex 1 (C1 - ) anion exchange resin. One of the procedures used to verify the product [1- 14 C]acetyl-L-carnitine can be used to synthesize (3S)-[5- 14 C]citric acid

  20. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism

    Science.gov (United States)

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu

    2015-01-01

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor α (PPARα) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH. PMID:26438600

  1. Comparative analysis of pharmacological treatments with N-acetyl-DL-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat.

    Science.gov (United States)

    Tighilet, Brahim; Leonard, Jacques; Bernard-Demanze, Laurence; Lacour, Michel

    2015-12-15

    Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

    Directory of Open Access Journals (Sweden)

    Clément Chevalier

    2010-03-01

    Full Text Available Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

  3. Succinyl-CoA:3-ketoacid CoA transferase (SCOT): cloning of the human SCOT gene, tertiary structural modeling of the human SCOT monomer, and characterization of three pathogenic mutations

    NARCIS (Netherlands)

    Fukao, T.; Mitchell, G. A.; Song, X. Q.; Nakamura, H.; Kassovska-Bratinova, S.; Orii, K. E.; Wraith, J. E.; Besley, G.; Wanders, R. J.; Niezen-Koning, K. E.; Berry, G. T.; Palmieri, M.; Kondo, N.

    2000-01-01

    The activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT; locus symbol OXCT; EC 2.8.3.5) is the main determinant of the ketolytic capacity of tissues. Hereditary SCOT deficiency causes episodic ketoacidosis. Here we describe the human SCOT gene, which spans more than 100 kb and contains 17

  4. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

    Directory of Open Access Journals (Sweden)

    Clemens Schmeitzl

    2015-08-01

    Full Text Available Deoxynivalenol (DON is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON, 15-acetyl-DON (15-ADON and 3,15-diacetyl-DON (3,15-diADON, and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G and of 15-acetyl-DON-3-sulfate (15-ADON3S as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G. This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

  5. Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis

    International Nuclear Information System (INIS)

    Shieh, J.; Whitman, W.B.

    1988-01-01

    To detect autotrophic CO 2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotropically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO 2 fixation was pulled in the direction of lactate synthesis, CO 2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO 2 and H 2 , but H 2 + CO 2 -independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min -1 mg of protein -1 . When BES was added, the rate of lactate synthesis increased to 2.1 nmol min -1 mg of protein -1 . Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14 CO 2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14 CH 2 O was specifically incorporated into the C-3 of lactate, and 14 CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO 2 assimilation

  6. Protein acetylation sites mediated by Schistosoma mansoni GCN5

    International Nuclear Information System (INIS)

    Moraes Maciel, Renata de; Furtado Madeiro da Costa, Rodrigo; Meirelles Bastosde Oliveira, Francisco; Rumjanek, Franklin David; Fantappie, Marcelo Rosado

    2008-01-01

    The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation

  7. Comparative Analysis of Proteome-Wide Lysine Acetylation in Juvenile and Adult Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    Qing Li

    2017-11-01

    Full Text Available Schistosomiasis is a devastating parasitic disease caused by tremotodes of the genus Schistosoma. Eggs produced by sexually mature schistosomes are the causative agents of for pathogenesis and transmission. Elucidating the molecular mechanism of schistosome development and sexual maturation would facilitate the prevention and control of schistosomiasis. Acetylation of lysine is a dynamic and reversible post-translational modification playing keys role in many biological processes including development in both eukaryotes and prokaryotes. To investigate the impacts of lysine acetylation on Schistosoma japonicum (S. japonicum development and sexual maturation, we used immunoaffinity-based acetyllysine peptide enrichment combined with mass spectrometry (MS, to perform the first comparative analysis of proteome-wide lysine acetylation in both female and male, juvenile (18 days post infection, 18 dpi and adult (28 dpi schistosome samples. In total, we identified 874 unique acetylated sites in 494 acetylated proteins. The four samples shared 47 acetylated sites and 46 proteins. More acetylated sites and proteins shared by both females and males were identified in 28 dpi adults (189 and 143, respectively than in 18 dpi schistosomula (76 and 59, respectively. More stage-unique acetylated sites and proteins were also identified in 28 dpi adults (494 and 210, respectively than in 18 dpi schistosomula (73 and 44, respectively. Functional annotation showed that in different developmental stages and genders, a number of proteins involving in muscle movement, glycometabolism, lipid metabolism, energy metabolism, environmental stress resistance, antioxidation, etc., displayed distinct acetylation profiles, which was in accordance with the changes of their biological functions during schistosome development, suggesting that lysine acetylation modification exerted important regulatory roles in schistosome development. Taken together, our data provided the first

  8. N-Acetyl-4-aminophenol (paracetamol), N-acetyl-2-aminophenol and acetanilide in urine samples from the general population, individuals exposed to aniline and paracetamol users.

    Science.gov (United States)

    Dierkes, Georg; Weiss, Tobias; Modick, Hendrik; Käfferlein, Heiko Udo; Brüning, Thomas; Koch, Holger M

    2014-01-01

    Epidemiological studies suggest associations between the use of N-acetyl-4-aminophenol (paracetamol) during pregnancy and increased risks of reproductive disorders in the male offspring. Previously we have reported a ubiquitous urinary excretion of N-acetyl-4-aminophenol in the general population. Possible sources are (1) direct intake of paracetamol through medication, (2) paracetamol residues in the food chain and (3) environmental exposure to aniline or related substances that are metabolized into N-acetyl-4-aminophenol. In order to elucidate the origins of the excretion of N-acetyl-4-aminophenol in urine and to contribute to the understanding of paracetamol and aniline metabolism in humans we developed a rapid, turbulent-flow HPLC-MS/MS method with isotope dilution for the simultaneous quantification of N-acetyl-4-aminophenol and two other aniline related metabolites, N-acetyl-2-aminophenol and acetanilide. We applied this method to three sets of urine samples: (1) individuals with no known exposure to aniline and also no recent paracetamol medication; (2) individuals after occupational exposure to aniline but no paracetamol medication and (3) paracetamol users. We confirmed the omnipresent excretion of N-acetyl-4-aminophenol. Additionally we revealed an omnipresent excretion of N-acetyl-2-aminophenol. In contrast, acetanilide was only found after occupational exposure to aniline, not in the general population or after paracetamol use. The results lead to four preliminary conclusions: (1) other sources than aniline seem to be responsible for the major part of urinary N-acetyl-4-aminophenol in the general population; (2) acetanilide is a metabolite of aniline in man and a valuable biomarker for aniline in occupational settings; (3) aniline baseline levels in the general population measured after chemical hydrolysis do not seem to originate from acetanilide and hence not from a direct exposure to aniline itself and (4) N-acetyl-2-aminophenol does not seem to be

  9. The Effect of Hypochlorite Oxidation and Acetylation on Some of the ...

    African Journals Online (AJOL)

    The study evaluated the effect of hypochlorite oxidation and acetylation on some physicochemical properties of Icacina trichantha starch. The native and modified (oxidized and acetylated) starches were studied with respect to Infrared spectroscopy(IR), microscopy, gelatinization, swelling power, solubility index, amylose ...

  10. Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

    International Nuclear Information System (INIS)

    Komoszynski, M.; Bandurski, R.S.

    1986-01-01

    Indole-3-acetyl-myo-inositol galactoside labeled with 3 H in the indole and 14 C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [ 3 H]indole-3-acetyl-myo-inositol and [ 3 H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumptions concerning the equilibration of applied [ 3 H]indole-3-acetyl-myo-inositol-[U- 14 C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indoleacetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [ 3 H]indole-3-acetyl-myo-inositol-[ 14 C] galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [ 3 H]indole-3-acetyl-myo-inositol-[ 14 C]galactose supplies appreciable amounts of 14 C to the shoot and both 14 C and 3 H to an uncharacterized insoluble fraction of the endosperm

  11. CPLA 1.0: an integrated database of protein lysine acetylation.

    Science.gov (United States)

    Liu, Zexian; Cao, Jun; Gao, Xinjiao; Zhou, Yanhong; Wen, Longping; Yang, Xiangjiao; Yao, Xuebiao; Ren, Jian; Xue, Yu

    2011-01-01

    As a reversible post-translational modification (PTM) discovered decades ago, protein lysine acetylation was known for its regulation of transcription through the modification of histones. Recent studies discovered that lysine acetylation targets broad substrates and especially plays an essential role in cellular metabolic regulation. Although acetylation is comparable with other major PTMs such as phosphorylation, an integrated resource still remains to be developed. In this work, we presented the compendium of protein lysine acetylation (CPLA) database for lysine acetylated substrates with their sites. From the scientific literature, we manually collected 7151 experimentally identified acetylation sites in 3311 targets. We statistically studied the regulatory roles of lysine acetylation by analyzing the Gene Ontology (GO) and InterPro annotations. Combined with protein-protein interaction information, we systematically discovered a potential human lysine acetylation network (HLAN) among histone acetyltransferases (HATs), substrates and histone deacetylases (HDACs). In particular, there are 1862 triplet relationships of HAT-substrate-HDAC retrieved from the HLAN, at least 13 of which were previously experimentally verified. The online services of CPLA database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0). The CPLA database is freely available for all users at: http://cpla.biocuckoo.org.

  12. A bioinformatics-based overview of protein Lys-Ne-acetylation

    Science.gov (United States)

    Among posttranslational modifications, there are some conceptual similarities between Lys-N'-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. T...

  13. A proteome-scale study on in vivo protein Nα-acetylation using an optimized method

    DEFF Research Database (Denmark)

    Zhang, Xumin; Ye, Juanying; Engholm-Keller, Kasper

    2011-01-01

    Protein N-terminal acetylation (N(α) -acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α) -modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to b...

  14. The Effect of Acetyl-L-Carnitine Administration on Persons with Down Syndrome

    Science.gov (United States)

    Pueschel, Siegfried M.

    2006-01-01

    Since previous investigations reported improvements in cognition of patients with dementia after acetyl-L-carnitine therapy and since there is an increased risk for persons with Down syndrome to develop Alzheimer disease, this study was designed to investigate the effect of acetyl-L-carnitine administration on neurological, intellectual, and…

  15. Protective Effects of Acetylation on the Pathological Reactions of the Lens Crystallins with Homocysteine Thiolactone.

    Directory of Open Access Journals (Sweden)

    Zeinab Moafian

    Full Text Available Various post-translational lens crystallins modifications result in structural and functional insults, contributing to the development of lens opacity and cataract disorders. Lens crystallins are potential targets of homocysteinylation, particularly under hyperhomocysteinemia which has been indicated in various eye diseases. Since both homocysteinylation and acetylation primarily occur on protein free amino groups, we applied different spectroscopic methods and gel mobility shift analysis to examine the possible preventive role of acetylation against homocysteinylation. Lens crystallins were extensively acetylated in the presence of acetic anhydride and then subjected to homocysteinylation in the presence of homocysteine thiolactone (HCTL. Extensive acetylation of the lens crystallins results in partial structural alteration and enhancement of their stability, as well as improvement of α-crystallin chaperone-like activity. In addition, acetylation partially prevents HCTL-induced structural alteration and aggregation of lens crystallins. Also, acetylation protects against HCTL-induced loss of α-crystallin chaperone activity. Additionally, subsequent acetylation and homocysteinylation cause significant proteolytic degradation of crystallins. Therefore, further experimentation is required in order to judge effectively the preventative role of acetylation on the structural and functional insults induced by homocysteinylation of lens crystallins.

  16. Prediction of Nepsilon-acetylation on internal lysines implemented in Bayesian Discriminant Method.

    Science.gov (United States)

    Li, Ao; Xue, Yu; Jin, Changjiang; Wang, Minghui; Yao, Xuebiao

    2006-12-01

    Protein acetylation is an important and reversible post-translational modification (PTM), and it governs a variety of cellular dynamics and plasticity. Experimental identification of acetylation sites is labor-intensive and often limited by the availability of reagents such as acetyl-specific antibodies and optimization of enzymatic reactions. Computational analyses may facilitate the identification of potential acetylation sites and provide insights into further experimentation. In this manuscript, we present a novel protein acetylation prediction program named PAIL, prediction of acetylation on internal lysines, implemented in a BDM (Bayesian Discriminant Method) algorithm. The accuracies of PAIL are 85.13%, 87.97%, and 89.21% at low, medium, and high thresholds, respectively. Both Jack-Knife validation and n-fold cross-validation have been performed to show that PAIL is accurate and robust. Taken together, we propose that PAIL is a novel predictor for identification of protein acetylation sites and may serve as an important tool to study the function of protein acetylation. PAIL has been implemented in PHP and is freely available on a web server at: http://bioinformatics.lcd-ustc.org/pail.

  17. Prediction of Nε-acetylation on internal lysines implemented in Bayesian Discriminant Method

    Science.gov (United States)

    Li, Ao; Xue, Yu; Jin, Changjiang; Wang, Minghui; Yao, Xuebiao

    2007-01-01

    Protein acetylation is an important and reversible post-translational modification (PTM), and it governs a variety of cellular dynamics and plasticity. Experimental identification of acetylation sites is labor-intensive and often limited by the availability reagents such as acetyl-specific antibodies and optimization of enzymatic reactions. Computational analyses may facilitate the identification of potential acetylation sites and provide insights into further experimentation. In this manuscript, we present a novel protein acetylation prediction program named PAIL, prediction of acetylation on internal lysines, implemented in a BDM (Bayesian Discriminant Method) algorithm. The accuracies of PAIL are 85.13%, 87.97% and 89.21% at low, medium and high thresholds, respectively. Both Jack-Knife validation and n-fold cross validation have been performed to show that PAIL is accurate and robust. Taken together, we propose that PAIL is a novel predictor for identification of protein acetylation sites and may serve as an important tool to study the function of protein acetylation. PAIL has been implemented in PHP and is freely available on a web server at: http://bioinformatics.lcd-ustc.org/pail. PMID:17045240

  18. Total levels of hippocampal histone acetylation predict normal variability in mouse behavior.

    Directory of Open Access Journals (Sweden)

    Addie May I Nesbitt

    Full Text Available Genetic, pharmacological, and environmental interventions that alter total levels of histone acetylation in specific brain regions can modulate behaviors and treatment responses. Efforts have been made to identify specific genes that are affected by alterations in total histone acetylation and to propose that such gene specific modulation could explain the effects of total histone acetylation levels on behavior - the implication being that under naturalistic conditions variability in histone acetylation occurs primarily around the promoters of specific genes.Here we challenge this hypothesis by demonstrating with a novel flow cytometry based technique that normal variability in open field exploration, a hippocampus-related behavior, was associated with total levels of histone acetylation in the hippocampus but not in other brain regions.Results suggest that modulation of total levels of histone acetylation may play a role in regulating biological processes. We speculate in the discussion that endogenous regulation of total levels of histone acetylation may be a mechanism through which organisms regulate cellular plasticity. Flow cytometry provides a useful approach to measure total levels of histone acetylation at the single cell level. Relating such information to behavioral measures and treatment responses could inform drug delivery strategies to target histone deacetylase inhibitors and other chromatin modulators to places where they may be of benefit while avoiding areas where correction is not needed and could be harmful.

  19. Histone Acetylation is Recruited in Consolidation as a Molecular Feature of Stronger Memories

    Science.gov (United States)

    Federman, Noel; Fustinana, Maria Sol; Romano, Arturo

    2009-01-01

    Gene expression is a key process for memory consolidation. Recently, the participation of epigenetic mechanisms like histone acetylation was evidenced in long-term memories. However, until now the training strength required and the persistence of the chromatin acetylation recruited are not well characterized. Here we studied whether histone…

  20. Multi-step rearrangement mechanism for acetyl cedrene to the hydrocarbon follower

    DEFF Research Database (Denmark)

    Paknikar, Shashikumar Keshav; Kamounah, Fadhil S.; Hansen, Poul Erik

    2017-01-01

    Conversion of acetyl cedrene (2) to its follower (3) using acetic anhydride and polyphosphoric acid involves a multi-step cationic molecular rearrangement, which is consistent with deuteriation and 1-13C labeling studies of acetyl cedrene. The key step involves cyclopropylcarbinyl cation-cyclopro...

  1. Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

    Science.gov (United States)

    Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

    2017-01-27

    The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogues

    Science.gov (United States)

    Lietzan, Adam D.; St. Maurice, Martin

    2014-01-01

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates. PMID:24157795

  3. H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells.

    Science.gov (United States)

    Ju, YoungJun; Untereiner, Ashley; Wu, Lingyun; Yang, Guangdong

    2015-11-01

    Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H(2)S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H(2)S S-sulfhydration is now proposed as a major mechanism for H(2)S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H(2)S and its implication in gluconeogenesis in the liver have been unknown. Gene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. Exogenously applied H(2)S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H(2)S-induced gluconeogenesis. H(2)S had no effect on the expressions of PC mRNA and protein, while H(2)S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H(2)S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H(2)S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. CSE/H(2)S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. Tissue-specific regulation of CSE/H(2)S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

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    Lin Chen

    2017-12-01

    Full Text Available Influenza A viruses (IAVs take advantage of the host acetylation system for their own benefit. Whether the nucleoprotein (NP of IAVs undergoes acetylation and the interaction between the NP and the class I histone deacetylases (HDACs were largely unknown. Here, we showed that the NP protein of IAV interacted with HDAC1, which downregulated the acetylation level of NP. Using mass spectrometry, we identified lysine 103 as an acetylation site of the NP. Compared with wild-type protein, two K103 NP mutants, K103A and K103R, enhanced replication efficiency of the recombinant viruses in vitro. We further demonstrated that HDAC1 facilitated viral replication via two paths: promoting the nuclear retention of NP and inhibiting TBK1-IRF3 pathway. Our results lead to a new mechanism for regulating NP acetylation, indicating that HDAC1 may be a possible target for antiviral drugs.

  5. Effect of Acetyl Group on Mechanical Properties of Chitin/Chitosan Nanocrystal: A Molecular Dynamics Study

    Directory of Open Access Journals (Sweden)

    Junhe Cui

    2016-01-01

    Full Text Available Chitin fiber is the load-bearing component in natural chitin-based materials. In these materials, chitin is always partially deacetylated to different levels, leading to diverse material properties. In order to understand how the acetyl group enhances the fracture resistance capability of chitin fiber, we constructed atomistic models of chitin with varied acetylation degree and analyzed the hydrogen bonding pattern, fracture, and stress-strain behavior of these models. We notice that the acetyl group can contribute to the formation of hydrogen bonds that can stabilize the crystalline structure. In addition, it is found that the specimen with a higher acetylation degree presents a greater resistance against fracture. This study describes the role of the functional group, acetyl groups, in crystalline chitin. Such information could provide preliminary understanding of nanomaterials when similar functional groups are encountered.

  6. Isolation and characterization of a thermolysin peptide containing acetyllysine from enzymatically acetylated f2al histone

    International Nuclear Information System (INIS)

    Horiuchi, Kentaro; Fujimoto, Daisaburo

    1973-01-01

    Previous studies (vol. 72, 433, '72) in this laboratory showed that histone acetylase in the cytosol of calf thymus introduced acetyl groups primarily into the epsilon-amino groups of lysine residues in a histone fraction, f2al. In an attempt to examine the site of acetylation in f2al by the enzyme, 14 C-acetylated f2al was isolated and digested by thermolysin. A radioactive peptide, which accounted for 50 - 60% of the total radioactivity, was obtained from the thermolysin digest and identified as the fragment containing amino acid residues 10-21. It appears, therefore, that the major sites of acetylation by the enzyme are the lysine 12 or 16 or both, which are known to be acetylated in vivo. It was also shown that the peptide was not deacetylated by histone deacetylase, in contrast with the whole f2al molecule. (author)

  7. Partially acetylated sugarcane bagasse for wicking oil from contaminated wetlands

    Energy Technology Data Exchange (ETDEWEB)

    Chung, S. [Samsung Engineering Co. Ltd., R and D Center, Suwon, Gyeonggi (Korea, Republic of); Suidan, M.T. [University of Cincinnati, School of Energy, Environmental, Biological and Medical Engineering, Cincinnati, OH (United States); Venosa, A.D. [NRMRL, U.S. EPA, Cincinnati, OH (United States)

    2011-12-15

    Sugarcane bagasse was partially acetylated to enhance its oil-wicking ability in saturated environments while holding moisture for hydrocarbon biodegradation. The water sorption capacity of raw bagasse was reduced fourfold after treatment, which indicated considerably increased hydrophobicity but not a limited capability to hold moisture for hydrocarbon biodegradation. Characterization results by Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and surface area analyzer suggested that treated bagasse exhibited enhanced hydrophobicity and surface area. Oil wicking test results indicate that treated bagasse is more effective in wicking oil from highly saturated environments than raw bagasse and suggest that application of this material in remediation of oil spills in highly saturated wetlands is promising. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  8. Acetylated Hyaluronic Acid: Enhanced Bioavailability and Biological Studies

    Directory of Open Access Journals (Sweden)

    Carmela Saturnino

    2014-01-01

    Full Text Available Hyaluronic acid (HA, a macropolysaccharidic component of the extracellular matrix, is common to most species and it is found in many sites of the human body, including skin and soft tissue. Not only does HA play a variety of roles in physiologic and in pathologic events, but it also has been extensively employed in cosmetic and skin-care products as drug delivery agent or for several biomedical applications. The most important limitations of HA are due to its short half-life and quick degradation in vivo and its consequently poor bioavailability. In the aim to overcome these difficulties, HA is generally subjected to several chemical changes. In this paper we obtained an acetylated form of HA with increased bioavailability with respect to the HA free form. Furthermore, an improved radical scavenging and anti-inflammatory activity has been evidenced, respectively, on ABTS radical cation and murine monocyte/macrophage cell lines (J774.A1.

  9. Acetylated flavonoid glycosides potentiating NGF action from Scoparia dulcis.

    Science.gov (United States)

    Li, Yushan; Chen, Xigui; Satake, Masayuki; Oshima, Yasukatsu; Ohizumi, Yasushi

    2004-04-01

    Three new acetylated flavonoid glycosides, 5,6,4'-trihydroxyflavone 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (1), apigenin 7-O-alpha-L-3-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (2), and apigenin 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (3), were isolated from Scoparia dulcis together with the known compound eugenyl beta-D-glucopyranoside (4). Their structures were elucidated by spectroscopic analyses. Compounds 2 and 3 showed an enhancing activity of nerve growth factor-mediated neurite outgrowth in PC12D cells.

  10. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    International Nuclear Information System (INIS)

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:α-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal α-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from [ 3 H]CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with [ 3 H]acetyl-CoA. The acetyl group can be transferred to glucosamine, forming [ 3 H]N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism

  11. Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay.

    Science.gov (United States)

    Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C J; Wallace, John C; Polyak, Steven W

    2008-11-15

    Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme's properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors.

  12. Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase

    DEFF Research Database (Denmark)

    Chen, Yun; Zhang, Yiming; Siewers, Verena

    2015-01-01

    Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported...

  13. Morphological, mechanical, barrier and properties of films based on acetylated starch and cellulose from barley.

    Science.gov (United States)

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Biduski, Bárbara; Evangelho, Jarine Amaral do; Bruni, Graziella Pinheiro; Antunes, Mariana Dias; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2017-01-01

    Biodegradable films of native or acetylated starches with different concentrations of cellulose fibers (0%, 10% and 20%) were prepared. The films were characterized by morphological, mechanical, barrier, and thermal properties. The tensile strength of the acetylated starch film was lower than those of the native starch film, without fibers. The addition of fibers increased the tensile strength and decreased the elongation and the moisture of native and acetylated starches films. The acetylated starch film showed higher water solubility when compared to native starch film. The addition of cellulose fibers reduced the water solubility of the acetylated starch film. The films reinforced with cellulose fiber exhibited a higher initial decomposition temperature and thermal stability. The mechanical, barrier, solubility, and thermal properties are factors which direct the type of the film application in packaging for food products. The films elaborated with acetylated starches of low degree of substitution were not effective in a reduction of the water vapor permeability. The addition of the cellulose fiber in acetylated and native starches films can contribute to the development of more resistant films to be applied in food systems that need to maintain their integrity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  14. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

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    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  15. Changes in nuclear protein acetylation in u. v. -damaged human cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramanathan, B.; Smerdon, M.J.

    1986-07-01

    We have investigated the levels of nuclear protein acetylation in u.v.-irradiated human fibroblasts. We measured the levels of acetylation in total acid-soluble nuclear proteins and observed two distinct differences between the irradiated and unirradiated (control) cells. Immediately after irradiation, there is a wave of protein hyperacetylation (i.e. a total acetylation level greater than that of unirradiated cells) that lasts for 2-6 h depending on the experimental conditions. This hyperacetylation phase is then followed by a hypoacetylation phase, lasting for many hours, and the total level of acetylation does not return to that of control cells until 24-72 h after u.v. damage. Both the magnitude and duration of each phase is dependent on the dose of u.v. light used. The wave of hyperacetylation is more pronounced at low u.v. doses (i.e. less than 5 J/m2), while the wave of hypoacetylation is more pronounced at higher u.v. doses (greater than or equal to 8 J/m2). Furthermore, the duration of each phase is prolonged when cells are exposed to 2 mM hydroxyurea. Examination of the acetylation levels of the individual nuclear proteins indicated that acetylation of the core histones follows the same pattern observed for the total acid-soluble protein fractions. Furthermore, these were the only major proteins in the total acid-soluble fraction observed to undergo the early, rapid hyperacetylation immediately following u.v. damage. Acetylation of histone H1 was negligible in both damaged and control cells, while three prominent non-histone proteins were acetylated only after long labeling times (greater than 4 h) in each case, gradually becoming hyperacetylated in the u.v.-damaged cells. These results raise the possibility that a causal relationship exists between nuclear protein acetylation and nucleotide excision repair of DNA in human cells.

  16. Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

    Science.gov (United States)

    Komoszynski, M.; Bandurski, R. S.

    1986-01-01

    Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

  17. Aspirin-Mediated Acetylation Protects Against Multiple Neurodegenerative Pathologies by Impeding Protein Aggregation.

    Science.gov (United States)

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Kakraba, Samuel; Alla, Ramani; Mehta, Jawahar L; Shmookler Reis, Robert J

    2017-12-10

    Many progressive neurological disorders, including Alzheimer's disease (AD), Huntington's disease, and Parkinson's disease (PD), are characterized by accumulation of insoluble protein aggregates. In prospective trials, the cyclooxygenase inhibitor aspirin (acetylsalicylic acid) reduced the risk of AD and PD, as well as cardiovascular events and many late-onset cancers. Considering the role played by protein hyperphosphorylation in aggregation and neurodegenerative diseases, and aspirin's known ability to donate acetyl groups, we asked whether aspirin might reduce both phosphorylation and aggregation by acetylating protein targets. Aspirin was substantially more effective than salicylate in reducing or delaying aggregation in human neuroblastoma cells grown in vitro, and in Caenorhabditis elegans models of human neurodegenerative diseases in vivo. Aspirin acetylates many proteins, while reducing phosphorylation, suggesting that acetylation may oppose phosphorylation. Surprisingly, acetylated proteins were largely excluded from compact aggregates. Molecular-dynamic simulations indicate that acetylation of amyloid peptide energetically disfavors its association into dimers and octamers, and oligomers that do form are less compact and stable than those comprising unacetylated peptides. Hyperphosphorylation predisposes certain proteins to aggregate (e.g., tau, α-synuclein, and transactive response DNA-binding protein 43 [TDP-43]), and it is a critical pathogenic marker in both cardiovascular and neurodegenerative diseases. We present novel evidence that acetylated proteins are underrepresented in protein aggregates, and that aggregation varies inversely with acetylation propensity after diverse genetic and pharmacologic interventions. These results are consistent with the hypothesis that aspirin inhibits protein aggregation and the ensuing toxicity of aggregates through its acetyl-donating activity. This mechanism may contribute to the neuro-protective, cardio

  18. Effect of glycolate on the activity of ribulosediphosphate and phosphoenolpyruvate carboxylases in C/sub 3/ and C/sub 4/ plants at different O/sub 2/ concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Popova, L; Dimitrova, O [Bylgarska Akademiya na Naukite, Sofia. Inst. po Fiziologiya na Rasteniyata

    1981-01-01

    The experiments are carried out with pisum and maize plants grown in a chamber under controlled conditions. The activity of the two enzymes is recorded by the amount of fixed H/sup 14/CO/sub 3/ in acid-stable products of the reactions. The effect of glycolate on the activity of the carboxylating enzymes is largely related to the effect of O/sub 2/ on the activity of the RuDP and PEP carboxylases. The stimulating effect of glucolate on the activity of the carboxylating enzymes is probably of a regulatory significance. An essential conclusion is that O/sub 2/ attacks C/sub 4/ photosynthesis not only on inhibiting the RuDP carboxylase in bundle sheath cells, but it also inhibits the activity of the main carboxylating enzyme in C/sub 4/ plants-the PEP carboxylase.

  19. The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

    International Nuclear Information System (INIS)

    Mu, J.-J.; Tsay, Y.-G.; Juan, L.-J.; Fu, T.-F.; Huang, W.-H.; Chen, D.-S.; Chen, P.-J.

    2004-01-01

    Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [ 3 H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication

  20. Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia.

    Science.gov (United States)

    Muñoz-Bertomeu, Jesús; Sales, Ester; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2007-11-01

    Spike lavender (Lavandula latifolia) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, beta-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T1 plants derived from self-pollinated seeds of T0 lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T0 plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species.

  1. Sudden unexpected infant death (SUDI in a newborn due to medium chain acyl CoA dehydrogenase (MCAD deficiency with an unusual severe genotype

    Directory of Open Access Journals (Sweden)

    Lovera Cristina

    2012-10-01

    Full Text Available Abstract Medium chain acyl CoA dehydrogenase deficiency (MCAD is the most common inborn error of fatty acid oxidation. This condition may lead to cellular energy shortage and cause severe clinical events such as hypoketotic hypoglycemia, Reye syndrome and sudden death. MCAD deficiency usually presents around three to six months of life, following catabolic stress as intercurrent infections or prolonged fasting, whilst neonatal-onset of the disease is quite rare. We report the case of an apparently healthy newborn who suddenly died at the third day of life, in which the diagnosis of MCAD deficiency was possible through peri-mortem blood-spot acylcarnitine analysis that showed very high concentrations of octanoylcarnitine. Genetic analysis at the ACADM locus confirmed the biochemical findings by demonstrating the presence in homozygosity of the frame-shift c.244dup1 (p.Trp82LeufsX23 mutation, a severe genotype that may explain the unusual and very early fatal outcome in this newborn. This report confirms that inborn errors of fatty acid oxidation represent one of the genetic causes of sudden unexpected deaths in infancy (SUDI and underlines the importance to include systematically specific metabolic screening in any neonatal unexpected death.

  2. Xenobiotic/medium chain fatty acid: CoA ligase - a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin.

    Science.gov (United States)

    van der Sluis, Rencia; Erasmus, Elardus

    2016-10-01

    Activation of fatty acids by the acyl-CoA synthetases (ACSs) is the vital first step in fatty acid metabolism. The enzymatic and physiological characterization of the human xenobiotic/medium chain fatty acid: CoA ligases (ACSMs) has been severely neglected even though xenobiotics, such as benzoate and salicylate, are detoxified through this pathway. This review will focus on the nomenclature and substrate specificity of the human ACSM ligases; the biochemical and enzymatic characterization of ACSM1 and ACSM2B; the high sequence homology of the ACSM2 genes (ACSM2A and ACSM2B) as well as what is currently known regarding disease association studies. Several discrepancies exist in the current literature that should be taken note of. For example, the single nucleotide polymorphisms (SNPs) reported to be associated with aspirin metabolism and multiple risk factors of metabolic syndrome are incorrect. Kinetic data on the substrate specificity of the human ACSM ligases are non-existent and currently no data exist on the influence of SNPs on the enzyme activity of these ligases. One of the biggest obstacles currently in the field is that glycine conjugation is continuously studied as a one-step process, which means that key regulatory factors of the two individual steps remain unknown.

  3. Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency: two pathogenic mutations, V133E and C456F, in Japanese siblings.

    Science.gov (United States)

    Song, X Q; Fukao, T; Watanabe, H; Shintaku, H; Hirayama, K; Kassovska-Bratinova, S; Kondo, N; Mitchell, G A

    1998-01-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5; locus symbol OXCT) is the key enzyme of ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We developed a transient expression system for mutant SCOT cDNAs, using immortalized SCOT-deficient fibroblasts. This paper describes and characterizes three missense mutations in two SCOT-deficient siblings from Japan. They are genetic compounds who inherited the mutation C456F (c1367 G-->T) from their mother. Their paternal allele contains two mutations in cis, T58M (c173 C-->T) and V133E (c398T-->A). Expression of SCOT cDNAs containing either V133E or C456F produces no detectable SCOT activity, whereas T58M is functionally neutral. T58M is a rare sequence variant not detected in 100 control Japanese alleles. In fibroblasts from the proband (GS02), in whom immunoblot demonstrated no detectable SCOT peptide, we measured an apparent residual SCOT activity of 20-35%. We hypothesize that the high residual SCOT activity in homogenates may be an artifact caused by use of the substrate, acetoacetyl-CoA by other enzymes. Expression of mutant SCOT cDNAs more accurately reflects the residual activity of SCOT than do currently available assays in cell or tissue homogenates.

  4. Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala.

    Science.gov (United States)

    Srivastava, Sameer; Vishwakarma, Rishi K; Arafat, Yasir Ali; Gupta, Sushim K; Khan, Bashir M

    2015-04-01

    Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.

  5. Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

    KAUST Repository

    Khraiwesh, Basel

    2013-05-13

    Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.

  6. Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

    KAUST Repository

    Khraiwesh, Basel; Harb, Jamil; Qudeimat, Enas

    2013-01-01

    Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.

  7. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

    DEFF Research Database (Denmark)

    Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

    2012-01-01

    Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

  8. Identification and characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Deborah M B Post

    Full Text Available Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA, which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as "AckA-dependent". Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for

  9. Inhibitory Effect on In Vitro LDL Oxidation and HMG Co-A Reductase Activity of the Liquid-Liquid Partitioned Fractions of Hericium erinaceus (Bull. Persoon (Lion’s Mane Mushroom

    Directory of Open Access Journals (Sweden)

    Mohammad Azizur Rahman

    2014-01-01

    Full Text Available Oxidation of low-density lipoprotein (LDL has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM, hexane (HEX, dichloromethane (DCM, ethyl acetate (EA, and aqueous residue (AQ. The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins for the formation of conjugated diene (CD at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL of thiobarbituric acid reactive substances (TBARS at 1 mg/mL. It also mostly inhibited (59.91% the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

  10. Inhibitory effect on in vitro LDL oxidation and HMG Co-A reductase activity of the liquid-liquid partitioned fractions of Hericium erinaceus (Bull.) Persoon (lion's mane mushroom).

    Science.gov (United States)

    Rahman, Mohammad Azizur; Abdullah, Noorlidah; Aminudin, Norhaniza

    2014-01-01

    Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

  11. Influence of the nitrate concentration and source in the incorporation of 14CO2 by the RuBP-carboxylase from wheat (triticum aestivum) and maize (zea mays)

    International Nuclear Information System (INIS)

    Saez Angulo, R.M.; Gines Diaz, M.J.; Garcia Pineda, M.D.

    1982-01-01

    The effect of the concentration and source of nitrogen in the culture media has been studied regarding its influence in the activity of the RuBP-carboxylase from wheat and maize during the first month of development. Wheat and maize has been chosen as plants representatives of two different types of CO 2 assimilation: C3 and M- respectively. Plants have been grown in hydroponic media and under temperature, humidity and nutrient salts control. A negative effect of NH 4 has been observed in the enzymatic activity of wheat seedlings, being this effect more remarkable as NH 4 concentration increases and as long the time of treatment. In our experimental conditions the most favorable source of nitrogen has been N0 3 NH 4 . The specific activity of the enzyme from wheat is about four times higher than in maize, even it decreases with time. This decreasing has not been observed in maize, with the exception of total absence of nitrogen in the media. We have not seen significant differences between the two photo periods which have been tested. Also, no differences have been found in the enzyme activities at the different NO 3 NH 4 concentrations assayed, and it seems that RuBP-carboxylase metabolism is only affected in the case of absolute stress. (Author) 20 refs

  12. Medium Density Fibreboard Made of Acetylated Sludge from Paper Mill

    Directory of Open Access Journals (Sweden)

    Luthfi Hakim

    2013-03-01

    Full Text Available Research of using sludge as raw material for making medium density fibreboard (MDF was useful to create additional value of sludge. The objective of the research was to evaluate physical properties, mechanical properties, and durability of MDF from acetylated sludge in 4 levels of acetate anhydride (0%, 3%, 5%, and 7% with 3 replicates. The MDF was made using dry process. After materials were mixed with adhesives, they were pressed using hotpress under 170 oC temperature and 45 Pa pressure for 25 minutes. The size of the MDF sample was 25 cm x 20 cm x 1 cm with 0.8 g/cm3 density. The physical properties (density, moisture content, water absorption, thickness swelling and mechanical properties (modulus of elasticity, modulus of rupture, internal bond, screw holding power was tested based on JIS A 5905-2003 standard. The durability was evaluated using SNI 01-7207-2006. All physical properties of MDF fulfill JIS A 5905-2003. Acetate anhydride decreased the moisture content value of MDF. On the other hand, all mechanical properties did not fulfill the standard. That was caused by calcium carbonate in sludge that blocked the adhesion between sludge fibres. The durability of MDF tested here was classified Class I which is very resistant to termites.

  13. Experimental thermochemical study of 3-acetyl-2-methyl-5-phenylthiophene

    International Nuclear Information System (INIS)

    Ribeiro da Silva, Manuel A.V.; Santos, Ana Filipa L.O.M.

    2010-01-01

    The standard (p 0 =0.1MPa) massic energy of combustion, in oxygen, of the crystalline 3-acetyl-2-methyl-5-phenylthiophene was measured, at T = 298.15 K, by rotating-bomb combustion calorimetry, from which the standard molar enthalpy of formation, in the condensed phase, was calculated as Δ f H m 0 (cr)=-(104.3±3.1)kJ.mol -1 . The corresponding standard molar enthalpy of sublimation, at T = 298.15 K, Δ cr g H m 0 =(108.9±0.4)kJ.mol -1 , was derived by the Clausius-Clapeyron equation, from the temperature dependence of the vapour pressures of this compound, measured by the Knudsen effusion mass-loss technique. From the results presented above, the standard molar enthalpy of formation, in the gaseous phase, at T = 298.15 K, was derived, Δ f H m 0 (g)=(4.6±3.1)kJ.mol -1 . This value, in conjunction with the literature values of the experimental enthalpies of formation of thiophene, 2-methylthiophene, and 3-acetylthiophene, was used to predict the enthalpic increment due to the introduction of a phenyl group in the position 2- of the thiophene ring. The calculated increment was compared with the corresponding ones in benzene and pyridine derivatives.

  14. Acetyl analogs of combretastatin A-4: synthesis and biological studies.

    Science.gov (United States)

    Babu, Balaji; Lee, Megan; Lee, Lauren; Strobel, Raymond; Brockway, Olivia; Nickols, Alexis; Sjoholm, Robert; Tzou, Samuel; Chavda, Sameer; Desta, Dereje; Fraley, Gregory; Siegfried, Adam; Pennington, William; Hartley, Rachel M; Westbrook, Cara; Mooberry, Susan L; Kiakos, Konstantinos; Hartley, John A; Lee, Moses

    2011-04-01

    The combretastatins have received significant attention because of their simple chemical structures, excellent antitumor efficacy and novel antivascular mechanisms of action. Herein, we report the synthesis of 20 novel acetyl analogs of CA-4 (1), synthesized from 3,4,5-trimethoxyphenylacetone that comprises the A ring of CA-4 with different aromatic aldehydes as the B ring. Molecular modeling studies indicate that these new compounds possess a 'twisted' conformation similar to CA-4. The new analogs effectively inhibit the growth of human and murine cancer cells. The most potent compounds 6k, 6s and 6t, have IC(50) values in the sub-μM range. Analog 6t has an IC(50) of 182 nM in MDA-MB-435 cells and has advantages over earlier analogs due to its enhanced water solubility (456 μM). This compound initiates microtubule depolymerization with an EC(50) value of 1.8 μM in A-10 cells. In a murine L1210 syngeneic tumor model 6t had antitumor activity and no apparent toxicity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Isolation of Acetyl Aleuritolic Acid from Pimeleodendron Griffithianum (Euphorbiaceae)

    International Nuclear Information System (INIS)

    Rosmawati Abdul Aziz; Norizan Ahmat; Rosmawati Abdul Aziz; Norizan Ahmat

    2016-01-01

    Pimeleodendron griffithianum, locally known as Perah ikan is available in Thailand, Peninsular Malaysia and Sumatra. The plant can be found in wet forest, secondary forest, pole forest, semi-swamps, and along logging roads or on hill slopes. In Malaysia, the stem of P. griffithianum being used as commercial timber. So far, no chemical constituents have been reported from P. griffithianum. In this study, a phytochemical study was conducted on the stem bark of P. griffithianum. The stem bark was obtained from Sungai Siput, Perak. The cleaned, chopped and dried stem-bark was extracted using acetone for several times. The separation of the components was carried out using vacuum liquid chromatography (VLC) followed by recrystallization method. The structure of the pure compound was elucidated by spectroscopic methods including nuclear magnetic resonance (NMR), ultraviolate - visible (UV-Vis) and comparison with literature. One compound (1) was isolated and identified as acetyl aleuritolic acid. This compound was reported for the first time from this plant. (author)

  16. In vivo labelling of acetyl-aspartyl peptides in mouse brain from intracranially and intracranially and intraperitoneally administered acetyl-L-[U-14C]aspartate

    International Nuclear Information System (INIS)

    Sinichkin, A.; Sterri, S.; Edminson, P.D.; Reichelt, K.L.; Kvamme, E.

    1977-01-01

    Following intracranial and intraperitoneal injection of acetyl-L-[U- 14 C]aspartate into mice about 5% and 0.7% of the radioactivity, respectively, was recovered from the brain after 30 min. On chromatographic separation of the cationic and anionic compounds on a Dowex 50 column, the former fraction contained about 60% of the radioactivity, predominantly as labelled asparate and glutamate. The anionic compounds, containing 20% of the labelled compounds, were fractionated in several chromatographic systems and resolved into a great variety of labelled peptidic compounds of which five acetyl-[U 14 ]aspartyl peptides, containing two to four amino acids, were purified. One of these, acetyl-aspartyl glutamine, has not previously been found in brain. (author)

  17. Acetylated rice starches films with different levels of amylose: Mechanical, water vapor barrier, thermal, and biodegradability properties.

    Science.gov (United States)

    Colussi, Rosana; Pinto, Vânia Zanella; El Halal, Shanise Lisie Mello; Biduski, Bárbara; Prietto, Luciana; Castilhos, Danilo Dufech; Zavareze, Elessandra da Rosa; Dias, Alvaro Renato Guerra

    2017-04-15

    Biodegradable films from native or acetylated starches with different amylose levels were prepared. The films were characterized according to the mechanical, water vapor barrier, thermal, and biodegradability properties. The films from acetylated high amylose starches had higher moisture content and water solubility than the native high amylose starch film. However, the acetylation did not affect acid solubility of the films, regardless of the amylose content. Films made from high and medium amylose rice starches were obtained; however low amylose rice starches, whether native or acetylated, did not form films with desirable characteristics. The acetylation decreased the tensile strength and increased the elongation of the films. The acetylated starch-based films had a lower decomposition temperature and higher thermal stability than native starch films. Acetylated starches films exhibited more rapid degradation as compared with the native starches films. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13).

    Science.gov (United States)

    Wallace, H M; Nuttall, M E; Robinson, F C

    1988-01-01

    Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine. PMID:3421945

  19. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Henriksen, Peter; Wagner, Sebastian Alexander; Weinert, Brian Tate

    2012-01-01

    Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine...... acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S....... cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved...

  20. An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

    Science.gov (United States)

    Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

    2015-12-07

    As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

  1. Identification and purification of O-acetyl-L-serine sulphhydrylase in Penicillium chrysogenum

    DEFF Research Database (Denmark)

    østergaard, Simon; Theilgaard, Hanne Birgitte; Nielsen, Jens Bredal

    1998-01-01

    We have demonstrated that Penicillium chrysogenum possesses the L-cysteine biosynthetic enzyme O-acetyI-L-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates...... the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-L-serine sulphhydrylase and O-acetyl-L-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-L-serine as substrate for the formation of L-cysteine....... The purified enzyme did not catalyse the formation of L-homocysteine from O-acetyl-L-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyI-L-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold...

  2. Maintenance of Glucose Homeostasis through Acetylation of the Metabolic Transcriptional Coactivator PGC-1alpha

    National Research Council Canada - National Science Library

    Puigserver, Pere

    2008-01-01

    The main purpose of this proposal is to test the hypothesis that acetylation of PGC-1alpha by GCN5 and associated proteins, Pc3 and WDR18, is a key regulatory modification that controls hepatic glucose production...

  3. Maintenance of Glucose Homeostasis through Acetylation of the Metabolic Transcriptional Coactivator PGC-1alpha

    National Research Council Canada - National Science Library

    Puigserver, Pere

    2007-01-01

    ... hepatic glucose production. This investigation has a define scope to specifically test how these proteins control the acetylation status of PGC-1alpha and what is the functional effect in blood glucose levels...

  4. Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

    International Nuclear Information System (INIS)

    Stefanovsky, V.Yu.; Dimitrov, S.I.; Angelov, D.; Pashev, I.G.

    1989-01-01

    The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA

  5. Monitoring the effect of belinostat in solid tumors by H4 acetylation

    DEFF Research Database (Denmark)

    Marquard, L.; Petersen, K.D.; Persson, M.

    2008-01-01

    after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin-embedded fine needle biopsies from nude...... acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors Udgivelsesdato: 2008/5...

  6. Peripheral effects of FAAH deficiency on fuel and energy homeostasis: role of dysregulated lysine acetylation.

    Directory of Open Access Journals (Sweden)

    Bhavapriya Vaitheesvaran

    Full Text Available FAAH (fatty acid amide hydrolase, primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA. Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/- mice.FAAH(-/- mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry. FAAH(-/- mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN. Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/- mice. Dysregulated hepatic FAAH(-/- lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/- acetyl-CoA (85%, p<0.01 corresponded to similar increases in citrate levels (45%. Altered FAAH(-/- mitochondrial malate dehydrogenase (MDH2 acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/- mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/- aldolase B. Fed FAAH(-/- alcohol dehydrogenase (ADH acetylation was also decreased.Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/- mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver

  7. N-acetyl Aspartate Levels in Adolescents With Bipolar and/or Cannabis Use Disorders

    Science.gov (United States)

    Bitter, Samantha M.; Weber, Wade A.; Chu, Wen-Jang; Adler, Caleb M.; Eliassen, James C.; Strakowski, Stephen M.; DelBello, Melissa P.

    2014-01-01

    Objective Bipolar and cannabis use disorders commonly co-occur during adolescence, and neurochemical studies may help clarify the pathophysiology underlying this co-occurrence. This study compared metabolite concentrations in the left ventral lateral prefrontal cortex among: adolescents with bipolar disorder (bipolar group; n=14), adolescents with a cannabis use disorder (cannabis use group, n=13), adolescents with cannabis use and bipolar disorders (bipolar and cannabis group, n=25), and healthy adolescents (healthy controls, n=15). We hypothesized that adolescents with bipolar disorder (with or without cannabis use disorder) would have decreased N-acetyl aspartate levels in the ventral lateral prefrontal cortex compared to the other groups, and that the bipolar and cannabis group would have the lowest N-acetyl aspartate levels of all groups. Methods N-acetyl aspartate concentrations in the left ventral lateral prefrontal cortex were obtained using Proton Magnetic Resonance Spectroscopy. Results Adolescents with bipolar disorder showed significantly lower left ventral lateral prefrontal cortex N-acetyl aspartate levels, but post-hoc analyses indicated that this was primarily due to increased N-acetyl aspartate levels in the cannabis group. The cannabis use disorder group had significantly higher N-acetyl aspartate levels compared to the bipolar disorder and the bipolar and cannabis groups (p=0.0002 and p=0.0002, respectively). Pearson correlations revealed a significant positive correlation between amount of cannabis used and N-acetyl aspartate concentrations. Conclusions Adolescents with cannabis use disorder showed higher levels of N-acetyl aspartate concentrations that were significantly positively associated with the amount of cannabis used; however, this finding was not present in adolescents with comorbid bipolar disorder. PMID:24729763

  8. The C2H3O+ chemi-ion acetyl cation or O-protonated ketene

    DEFF Research Database (Denmark)

    Egsgaard, H.; Carlsen, L.

    1995-01-01

    The C2H3O+ chemi-ion sampled from a premixed methane/oxygen flame has been demonstrated to be the acetyl cation based on ion-molecule reactions with isoprene and 1,3-dioxolane.......The C2H3O+ chemi-ion sampled from a premixed methane/oxygen flame has been demonstrated to be the acetyl cation based on ion-molecule reactions with isoprene and 1,3-dioxolane....

  9. Targeted amino-terminal acetylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Matthew Johnson

    2010-12-01

    Full Text Available One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.

  10. Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

    Science.gov (United States)

    Pawar, Prashant Mohan-Anupama; Ratke, Christine; Balasubramanian, Vimal K; Chong, Sun-Li; Gandla, Madhavi Latha; Adriasola, Mathilda; Sparrman, Tobias; Hedenström, Mattias; Szwaj, Klaudia; Derba-Maceluch, Marta; Gaertner, Cyril; Mouille, Gregory; Ezcurra, Ines; Tenkanen, Maija; Jönsson, Leif J; Mellerowicz, Ewa J

    2017-06-01

    High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  11. Changes in nuclear protein acetylation in u.v.-damaged human cells

    International Nuclear Information System (INIS)

    Ramanathan, B.; Smerdon, M.J.

    1986-01-01

    The levels of nuclear protein acetylation in u.v.-irradiated human fibroblasts have been investigated. Initially, we measured the levels of acetylation in total acid-soluble nuclear proteins and observed two distinct differences between the irradiated and unirradiated (control) cells. Immediately after irradiation, there is a 'wave' of protein hyperacetylation that lasts for 2-6 h, followed by a hypoacetylation phase, lasting for many hours, and the total level of acetylation does not return to that of control cells until 24-72 h after u.v. damage. Both the magnitude and duration of each phase is dependent on the dose of u.v. light used. The wave of hyperacetylation is more pronounced at low u.v. doses, while the wave of hypoacetylation is more pronounced at higher u.v. doses. Furthermore, the duration of each phase is prolonged when cells are exposed to 2 mM hydroxyurea, an agent which retards the rate of excision repair at u.v.-damaged sites. Examinations of the acetylation levels of the individual nuclear proteins indicated that acetylation of the core histones follows the same pattern observed for the total acid-soluble protein fractions. Furthermore, these were the only major proteins in the total acid-soluble fraction observed to undergo the early, rapid hyperacetylation immediately following u.v. damage. These results raise the possibility that a causal relationship exists between nuclear protein acetylation and nucleotide excision repair of DNA in human cells. (author)

  12. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pathak, Ravi [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Philizaire, Marc [Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States); Mujtaba, Shiraz, E-mail: smujtaba@mec.cuny.edu [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States)

    2015-08-19

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets.

  13. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Pathak, Ravi; Philizaire, Marc; Mujtaba, Shiraz

    2015-01-01

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets

  14. Application of the MIDAS approach for analysis of lysine acetylation sites.

    Science.gov (United States)

    Evans, Caroline A; Griffiths, John R; Unwin, Richard D; Whetton, Anthony D; Corfe, Bernard M

    2013-01-01

    Multiple Reaction Monitoring Initiated Detection and Sequencing (MIDAS™) is a mass spectrometry-based technique for the detection and characterization of specific post-translational modifications (Unwin et al. 4:1134-1144, 2005), for example acetylated lysine residues (Griffiths et al. 18:1423-1428, 2007). The MIDAS™ technique has application for discovery and analysis of acetylation sites. It is a hypothesis-driven approach that requires a priori knowledge of the primary sequence of the target protein and a proteolytic digest of this protein. MIDAS essentially performs a targeted search for the presence of modified, for example acetylated, peptides. The detection is based on the combination of the predicted molecular weight (measured as mass-charge ratio) of the acetylated proteolytic peptide and a diagnostic fragment (product ion of m/z 126.1), which is generated by specific fragmentation of acetylated peptides during collision induced dissociation performed in tandem mass spectrometry (MS) analysis. Sequence information is subsequently obtained which enables acetylation site assignment. The technique of MIDAS was later trademarked by ABSciex for targeted protein analysis where an MRM scan is combined with full MS/MS product ion scan to enable sequence confirmation.

  15. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

    Directory of Open Access Journals (Sweden)

    Todd J Cohen

    Full Text Available Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD. Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

  16. Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis

    Science.gov (United States)

    Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L.; Shen, Yan; Qiu, Xiao-Bo

    2013-01-01

    SUMMARY Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes (“spermatoproteasomes”) contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739

  17. Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

    Directory of Open Access Journals (Sweden)

    Ferah Yildirim

    Full Text Available Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT and deacetylase activities (HDAC. Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB-binding protein (CBP as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min subthreshold occlusion of the middle cerebral artery (MCA, followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

  18. Comparative genomics and proteomics of vertebrate diacylglycerol acyltransferase (DGAT), acyl CoA wax alcohol acyltransferase (AWAT) and monoacylglycerol acyltransferase (MGAT).

    Science.gov (United States)

    Holmes, Roger S

    2010-03-01

    BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the acylglycerol acyltransferase (AGAT) superfamily. Evidence is reported for 8 opossum monoacylglycerol acyltransferase-like (MGAT) (E.C. 2.3.1.22) and diacylglycerol acyltransferase-like (DGAT) (E.C. 2.3.1.20) genes and proteins, including DGAT1, DGAT2, DGAT2L6 (DGAT2-like protein 6), AWAT1 (acyl CoA wax alcohol acyltransferase 1), AWAT2, MGAT1, MGAT2 and MGAT3. Three of these genes (AWAT1, AWAT2 and DGAT2L6) are closely localized on the opossum X chromosome. Evidence is also reported for six zebrafish MGAT- and DGAT-like genes, including two DGAT1-like genes, as well as DGAT2-, MGAT1-, MGAT2- and MGAT3-like genes and proteins. Predicted primary, secondary and transmembrane structures for the opossum and zebrafish MGAT-, AWAT- and DGAT-like subunits and the intron-exon boundaries for genes encoding these enzymes showed a high degree of similarity with other members of the AGAT superfamily, which play major roles in triacylglyceride (DGAT), diacylglyceride (MGAT) and wax ester (AWAT) biosynthesis. Alignments of predicted opossum, zebrafish and other vertebrate DGAT1, DGAT2, other DGAT2-like and MGAT-like amino acid sequences with known human and mouse enzymes demonstrated conservation of residues which are likely to play key roles in catalysis, lipid binding or in maintaining structure. Phylogeny studies of the human, mouse, opossum, zebrafish and pufferfish MGAT- and DGAT-like enzymes indicated that the common ancestors for these genes predated the appearance of bony fish during vertebrate evolution whereas the AWAT- and DGAT2L6-like genes may have appeared more recently prior to the appearance of marsupial and eutherian mammals. Copyright 2009 Elsevier Inc. All rights reserved.

  19. A pharmacologic increase in activity of plasma transaminase derived from small intestine in animals receiving an acyl CoA: diacylglycerol transferase (DGAT) 1 inhibitor.

    Science.gov (United States)

    Yokoyama, Hideaki; Kobayashi, Akio; Kondo, Kazuma; Oshida, Shin-Ichi; Takahashi, Tadakazu; Masuyama, Taku; Shoda, Toshiyuki; Sugai, Shoichiro

    2018-01-01

    Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the mechanism of the increase in transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. After dosing JTT-553, transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was due to increased enzyme protein synthesis and was suppressed by inhibiting fatty acid-transport to the enterocytes. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to release of the enzymes from injured cells into the circulation but to be phenomena resulting from enhancement of enzyme protein synthesis in the small intestine due to the pharmacological action of JTT-553 in this organ.

  20. Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Huanan; Song, Zhihong; Nikolau, Basil J.

    2012-03-31

    Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT‐encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T‐DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl CoA precursor required for the cytosol‐localized, mevalonate‐derived isoprenoid biosynthetic pathway.

  1. Amino acid solutions for premature neonates during the first week of life: the role of N-acetyl-L-cysteine and N-acetyl-L-tyrosine

    NARCIS (Netherlands)

    van Goudoever, J. B.; Sulkers, E. J.; Timmerman, M.; Huijmans, J. G.; Langer, K.; Carnielli, V. P.; Sauer, P. J.

    1994-01-01

    Tyrosine and cyst(e)ine are amino acids that are thought to be essential for preterm neonates. These amino acids have low stability (cyst(e)ine) or low solubility (tyrosine) and are therefore usually present only in small amounts in amino acid solutions. Acetylation improves the stability and

  2. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose.

    Science.gov (United States)

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Petrescu, Anca D; Landrock, Kerstin K; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

  3. Nitric Oxide Modulates Histone Acetylation at Stress Genes by Inhibition of Histone Deacetylases1[OPEN

    Science.gov (United States)

    Mengel, Alexander; Ageeva, Alexandra; Durner, Jörg

    2017-01-01

    Histone acetylation, which is an important mechanism to regulate gene expression, is controlled by the opposing action of histone acetyltransferases and histone deacetylases (HDACs). In animals, several HDACs are subjected to regulation by nitric oxide (NO); in plants, however, it is unknown whether NO affects histone acetylation. We found that treatment with the physiological NO donor S-nitrosoglutathione (GSNO) increased the abundance of several histone acetylation marks in Arabidopsis (Arabidopsis thaliana), which was strongly diminished in the presence of the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. This increase was likely triggered by NO-dependent inhibition of HDAC activity, since GSNO and S-nitroso-N-acetyl-dl-penicillamine significantly and reversibly reduced total HDAC activity in vitro (in nuclear extracts) and in vivo (in protoplasts). Next, genome-wide H3K9/14ac profiles in Arabidopsis seedlings were generated by chromatin immunoprecipitation sequencing, and changes induced by GSNO, GSNO/2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or trichostatin A (an HDAC inhibitor) were quantified, thereby identifying genes that display putative NO-regulated histone acetylation. Functional classification of these genes revealed that many of them are involved in the plant defense response and the abiotic stress response. Furthermore, salicylic acid, which is the major plant defense hormone against biotrophic pathogens, inhibited HDAC activity and increased histone acetylation by inducing endogenous NO production. These data suggest that NO affects histone acetylation by targeting and inhibiting HDAC complexes, resulting in the hyperacetylation of specific genes. This mechanism might operate in the plant stress response by facilitating the stress-induced transcription of genes. PMID:27980017

  4. Nitric Oxide Modulates Histone Acetylation at Stress Genes by Inhibition of Histone Deacetylases.

    Science.gov (United States)

    Mengel, Alexander; Ageeva, Alexandra; Georgii, Elisabeth; Bernhardt, Jörg; Wu, Keqiang; Durner, Jörg; Lindermayr, Christian

    2017-02-01

    Histone acetylation, which is an important mechanism to regulate gene expression, is controlled by the opposing action of histone acetyltransferases and histone deacetylases (HDACs). In animals, several HDACs are subjected to regulation by nitric oxide (NO); in plants, however, it is unknown whether NO affects histone acetylation. We found that treatment with the physiological NO donor S-nitrosoglutathione (GSNO) increased the abundance of several histone acetylation marks in Arabidopsis (Arabidopsis thaliana), which was strongly diminished in the presence of the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. This increase was likely triggered by NO-dependent inhibition of HDAC activity, since GSNO and S-nitroso-N-acetyl-dl-penicillamine significantly and reversibly reduced total HDAC activity in vitro (in nuclear extracts) and in vivo (in protoplasts). Next, genome-wide H3K9/14ac profiles in Arabidopsis seedlings were generated by chromatin immunoprecipitation sequencing, and changes induced by GSNO, GSNO/2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or trichostatin A (an HDAC inhibitor) were quantified, thereby identifying genes that display putative NO-regulated histone acetylation. Functional classification of these genes revealed that many of them are involved in the plant defense response and the abiotic stress response. Furthermore, salicylic acid, which is the major plant defense hormone against biotrophic pathogens, inhibited HDAC activity and increased histone acetylation by inducing endogenous NO production. These data suggest that NO affects histone acetylation by targeting and inhibiting HDAC complexes, resulting in the hyperacetylation of specific genes. This mechanism might operate in the plant stress response by facilitating the stress-induced transcription of genes. © 2017 American Society of Plant Biologists. All Rights Reserved.

  5. PSG gene expression is up-regulated by lysine acetylation involving histone and nonhistone proteins.

    Directory of Open Access Journals (Sweden)

    Soledad A Camolotto

    Full Text Available BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140, was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA. This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.

  6. Phytochrome control of gene expression in radish seedlings. 111. Evidence for a rapid control of the ribulose 1. 5 biphosphate carboxylase small subunit gene expression by red light

    Energy Technology Data Exchange (ETDEWEB)

    Fourcroy, P

    1986-01-01

    The effect of red and far-red light on the level of the mRNA encoding the small subunit (SSU) of ribulose, 1.5 bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) from radish cotyledons was investigated. Northern blot analysis of RNA with a cDNA probe showed that both long (12-36h) far-red irradiation and short (1-5 min) red irradiation brings about an increase in SSU mRNA concentraton which was prevented by a subsequent far-red light exposure. Far-red light was effective in reversing the red light effect provided that it was given soon after (<10 min) the red light pulse. The red light mediated increase in SSU mRNA level did not occur in presence of ..cap alpha..-amanitin. Our results suggest that phytochrome control of SSU gene expression is exerted at the transcriptional level. 34 refs.

  7. Photoperiodism and crassulacean acid metabolism : I. Immunological and kinetic evidences for different patterns of phosphoenolpyruvate carboxylase isoforms in photoperiodically inducible and non-inducible Crassulacean acid metabolism plants.

    Science.gov (United States)

    Brulfert, J; Müller, D; Kluge, M; Queiroz, O

    1982-05-01

    Plants of Kalanchoe blossfeldiana v. Poelln. Tom Thumb and Sedum morganianum E. Walth. were grown under controlled photoperiodic conditions under either short or long days. Gaz exchange measurements confirmed that in K. blossfeldiana Crassulacean acid metabolism (CAM) was photoperiodically inducible and that S. morganianum performed CAM independently of photoperiod. With K. blossfeldiana, a comparison of catalytic and regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from short-day and long-day grown plants showed differences, but not with S. morganianum. Ouchterlony double diffusion tests and immunotitration experiments (using a S. morganianum PEPC antibody) established that CAM is induced in K. blossfeldiana-but not in S. morganianum-through the synthesis of a new PEPC isoform; this form shows an immunological behavior different from that prevailing under non-inductive conditions and can be considered as specific for CAM performance.

  8. Behavioral neuroadaptation to alcohol : from glucocorticoids to histone acetylation

    Directory of Open Access Journals (Sweden)

    Daniel Beracochea

    2016-10-01

    mediating working memory impairments and neuroadaptive changes during withdrawal from chronic alcohol intake. It then highlights the role of cAMP-PKA-CREB signaling cascade and histone acetylation within the prefrontal cortex and limbic structures in alcohol-induced anxiety and behavioral impairments, and how an understanding of functional alterations of these pathways might lead to better treatments for neuropsychiatric disorders.

  9. Effect of acetylation on monoclonal antibody ZCE-025 Fab': Distribution in normal and tumor-bearing mice

    International Nuclear Information System (INIS)

    Tarburton, J.P.; Halpern, S.E.; Hagan, P.L.; Sudora, E.; Chen, A.; Fridman, D.M.; Pfaff, A.E.

    1990-01-01

    Studies were performed to determine in vitro and in vivo effects of acetylation on Fab' fragments of ZCE-025, a monoclonal anti-CEA antibody. Isoelectric focusing revealed a drop in isoelectric point of 1.7 pI units following acetylation. Biodistribution studies of acetylated and nonacetylated [111In]Fab' were performed in normal BALB/c mice and in nude mice bearing the T-380 CEA-producing human colon tumor. The acetylated fragments remained in the vascular compartment longer and had significantly diminished renal uptake of 111In compared to controls. While acetylation itself effected a 50% drop in immunoreactivity, tumor uptake of the acetylated and nonacetylated 111In-labeled Fab' fragments was comparable, with the exception of one data point, through 72 h

  10. Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids.

    Science.gov (United States)

    Oliva, R; Mezquita, C

    1982-01-01

    In order to study the relationship between acetylation of histones, chromatin structure and gene activity, the distribution and turnover of acetyl groups among nucleosomal core histones and the extent of histone H4 acetylation were examined in rooster testis cell nuclei at different stages of spermatogenesis. Histone H4 was the predominant acetylated histone in mature testes. Hyperacetylation of H4 and rapid turnover of its acetyl groups are not univocally correlated with transcriptional activity since they were detected in both genetically active testicular cells and genetically inactive elongated spermatids. During the transition from nucleohistone to nucleoprotamine in elongated spermatids the chromatin undergoes dramatic structural changes with exposition of binding sites on DNA (1). Hyperacetylation of H4 and rapid turnover of its acetyl groups could be correlated with the particular conformation of chromatin in elongated spermatids and might represent a necessary condition for binding of chromosomal proteins to DNA. Images PMID:7162988

  11. Effect of acetylation on antioxidant and cytoprotective activity of polysaccharides isolated from pumpkin (Cucurbita pepo, lady godiva).

    Science.gov (United States)

    Song, Yi; Yang, Yang; Zhang, Yuyu; Duan, Liusheng; Zhou, Chunli; Ni, Yuanying; Liao, Xiaojun; Li, Quanhong; Hu, Xiaosong

    2013-10-15

    Acetylation of pumpkin (Cucurbita pepo, lady godiva variety) polysaccharide using acetic anhydride with pyridines as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale. Furthermore, antioxidant activities and cytoprotective effects of pumpkin polysaccharide and its acetylated derivatives were investigated employing various established in vitro systems. Results showed that addition of pyridine as catalyst could increase the degree of substitution, whereas volume of acetic anhydride had little effect. The acetylated polysaccharides in DPPH scavenging radical activity assay, superoxide anion radical activity assay and reducing power assay exhibited higher antioxidant activity than that of unmodified polysaccharide. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by pumpkin polysaccharide and its acetylated derivatives and the derivatives presented higher protective effects. On the whole, acetylated polysaccharide showed relevant antioxidant activity both in vitro and in a cell system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Photoperiodism and Crassulacean acid metabolism : III. Different characteristics of the photoperiod-sensitive and non-sensitive isoforms of phosphoenolpyruvate carboxylase and Crassulacean acid metabolism operation.

    Science.gov (United States)

    Brulfert, J; Queiroz, O

    1982-05-01

    Sensitivity to glucose-6-P, inorganic phosphate and malate, Km phosphoenolpyruvate (PEP), and the effect of pH were comparatively investigated for phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) extracted along the day-night cycle from leaves of Kalanchoe blossfeldiana v. Poelln. Tom Thumb, grown under different photoperiodic conditions. Under long days, sensitivity to effectors is weak and varies only slightly during the 24h cycle, together with small variations of Km (PEP), ranging from 1.2 to 1.8 mM. The response-curve to pH shows two peaks for pH 7.4 and 8.4. Transfer of the plants to short days established an increase in the sensitivity of the enzyme to the effectors together with the appearance of a day-night variation of this effect, maximum during the day. A clear diurnal oscillation of Km (PEP) is observed from 3.1 mM at the beginning of day (09.00 h) to 0.9 mM at midnight (00.00 h), at pH 7.4. These results complement previous electrophoretic and immunological data by affording enzyme kinetic evidence that short days induce a PEP carboxylase form responsible for full-Crassulacean acid metabolism (CAM) operation which is kinetically different from the photoperiod-sensitive, C3-like form prevailing in young leaves under long days. The results indicate that diurnal enzymic rhythms would improve the efficiency of the metabolic regulatory mechanisms and act as a coordinating factor for the daily and seasonal adaptive operation of CAM.

  13. Preliminary study for acetylation of cassava bagasse starch and microfibrillated cellulose of bamboo

    Directory of Open Access Journals (Sweden)

    Silviana Silviana

    2018-01-01

    Full Text Available Bio composite matrixes have been developed from several biomaterials, such as starch. One of potential resources is starch isolated from cassava bagasse still consisting 30-50% of starch. Reinforcement material may be inserted into bio composite to tough and reduce the drawback of the starch-based bio composite or bio plastic. Microfibrillated cellulose of bamboo (MFC can be used as toughening filler for composite matrix. However, surface modification of material could be employed to alter its properties, such as acetylation of starch-based bio composite and microfibrillated cellulose. The acetylation was executed by using glacial acetic acid (GAA catalyzed with sodium hydroxide. This paper investigates optimum condition of acetylation for bagasse starch (BS and bamboo MFC in different weight ratio of GAA to BS or MFC (1:1, 2:1, 3:1, 1:2, 1:3, temperature range of 30°C to 70°C, and pH range of 7 to 11. Data were resulted from degree of susbtitution for each running. The optimum condition of acetylation of BS was obtained at temperature of 50°C (for BS and 30°C (for MFC, pH of 9, and 2:1 ratio. This acetylation was confirmed by fourier transform infrared spectroscopy and scanning electron microscope.

  14. PCAF/GCN5-Mediated Acetylation of RPA1 Promotes Nucleotide Excision Repair

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    Meimei Zhao

    2017-08-01

    Full Text Available The RPA complex can integrate multiple stress signals into diverse responses by activating distinct DNA repair pathways. However, it remains unclear how RPA1 elects to activate a specific repair pathway during different types of DNA damage. Here, we report that PCAF/GCN5-mediated K163 acetylation of RPA1 is crucial for nucleotide excision repair (NER but is dispensable for other DNA repair pathways. Mechanistically, we demonstrate that the acetylation of RPA1 is critical for the steady accumulation of XPA at damaged DNA sites and preferentially activates the NER pathway. DNA-PK phosphorylates and activates PCAF upon UV damage and consequently promotes the acetylation of RPA1. Moreover, the acetylation of RPA1 is tightly regulated by HDAC6 and SIRT1. Together, our results demonstrate that the K163 acetylation of RPA1 plays a key role in the repair of UV-induced DNA damage and reveal how the specific RPA1 modification modulates the choice of distinct DNA repair pathways.

  15. Inhibition of p53 acetylation by INHAT subunit SET/TAF-Iβ represses p53 activity.

    Science.gov (United States)

    Kim, Ji-Young; Lee, Kyu-Sun; Seol, Jin-Ee; Yu, Kweon; Chakravarti, Debabrata; Seo, Sang-Beom

    2012-01-01

    The tumor suppressor p53 responds to a wide variety of cellular stress signals. Among potential regulatory pathways, post-translational modifications such as acetylation by CBP/p300 and PCAF have been suggested for modulation of p53 activity. However, exactly how p53 acetylation is modulated remains poorly understood. Here, we found that SET/TAF-Iβ inhibited p300- and PCAF-mediated p53 acetylation in an INHAT (inhibitor of histone acetyltransferase) domain-dependent manner. SET/TAF-Iβ interacted with p53 and repressed transcription of p53 target genes. Consequently, SET/TAF-Iβ blocked both p53-mediated cell cycle arrest and apoptosis in response to cellular stress. Using different apoptosis analyses, including FACS, TUNEL and BrdU incorporation assays, we also found that SET/TAF-Iβ induced cellular proliferation via inhibition of p53 acetylation. Furthermore, we observed that apoptotic Drosophila eye phenotype induced by either dp53 overexpression or UV irradiation was rescued by expression of dSet. Inhibition of dp53 acetylation by dSet was observed in both cases. Our findings provide new insights into the regulation of stress-induced p53 activation by HAT-inhibiting histone chaperone SET/TAF-Iβ.

  16. Asymmetric distribution of glucose and indole-3-acetyl-myo-inositol in geostimulated Zea mays seedlings

    Science.gov (United States)

    Momonoki, Y. S.; Bandurski, R. S. (Principal Investigator)

    1988-01-01

    Indole-3-acetyl-myo-inositol occurs in both the kernel and vegetative shoot of germinating Zea mays seedlings. The effect of a gravitational stimulus on the transport of [3H]-5-indole-3-acetyl-myo-inositol and [U-14C]-D-glucose from the kernel to the seedling shoot was studied. Both labeled glucose and labeled indole-3-acetyl-myo-inositol become asymmetrically distributed in the mesocotyl cortex of the shoot with more radioactivity occurring in the bottom half of a horizontally placed seedling. Asymmetric distribution of [3H]indole-3-acetic acid, derived from the applied [3H]indole-3-acetyl-myo-inositol, occurred more rapidly than distribution of total 3H-radioactivity. These findings demonstrate that the gravitational stimulus can induce an asymmetric distribution of substances being transported from kernel to shoot. They also indicate that, in addition to the transport asymmetry, gravity affects the steady state amount of indole-3-acetic acid derived from indole-3-acetyl-myo-inositol.

  17. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  18. The Caenorhabditis elegans Elongator complex regulates neuronal alpha-tubulin acetylation.

    Directory of Open Access Journals (Sweden)

    Jachen A Solinger

    2010-01-01

    Full Text Available Although acetylated alpha-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate alpha-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of alpha-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of alpha-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating alpha-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3 and in a scaffold subunit (Elp1 have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

  19. Radiolysis of N-acetyl amino acids as model compounds for radiation degradation of polypeptides

    International Nuclear Information System (INIS)

    Garrett, R.W.; Hill, D.J.T.; Ho, S.Y.; O'Donnell, J.H.; O'Sullivan, P.W.; Pomery, P.J.

    1982-01-01

    Radiation chemical yields of (i) the volatile radiolysis products and (ii) the trapped free radicals from the γ-radiolysis of the N-acetyl derivatives of glycine, L-alanine, L-valine, L-phenylalanine and L-tyrosine in the polycrystalline state have been determined at room temperature (303 K). Carbon dioxide was found to be the major molecular product for all these compounds with G(CO 2 ) varying from 0.36 for N-acetyl-L-tyrosine to 8 for N-acetyl-L-valine. There was evidence for some scission of the N-Csub(α) bond, indicated by the production of acetamide and the corresponding aliphatic acid, but the deamination reaction was found to be of much lesser importance than the decarboxylation reaction. A protective effect of the aromatic ring in N-acetyl-L-phenylalanine and in N-acetyl-L-tyrosine was indicated by the lower yields of volatile products for these compounds. The yields of trapped free radicals were found to vary with the nature of the amino acid side chain, increasing with chain length and chain branching. The radical yields were decreased by incorporation of an aromatic moiety in the side chain, this effect being greater for the tyrosyl side chain than for the phenyl side chain. The G(R) values showed a good correlation with G(CO 2 ) indicating that a common reaction may be involved in radical production and carbon dioxide formation. (author)

  20. Mechanism of host substrate acetylation by a YopJ family effector.

    Science.gov (United States)

    Zhang, Zhi-Min; Ma, Ka-Wai; Gao, Linfeng; Hu, Zhenquan; Schwizer, Simon; Ma, Wenbo; Song, Jikui

    2017-07-24

    The Yersinia outer protein J (YopJ) family of bacterial effectors depends on a novel acetyltransferase domain to acetylate signalling proteins from plant and animal hosts. However, the underlying mechanism is unclear. Here, we report the crystal structures of PopP2, a YopJ effector produced by the plant pathogen Ralstonia solanacearum, in complex with inositol hexaphosphate (InsP 6 ), acetyl-coenzyme A (AcCoA) and/or substrate Resistance to Ralstonia solanacearum 1 (RRS1-R) WRKY . PopP2 recognizes the WRKYGQK motif of RRS1-R WRKY to position a targeted lysine in the active site for acetylation. Importantly, the PopP2-RRS1-R WRKY association is allosterically regulated by InsP 6 binding, suggesting a previously unidentified role of the eukaryote-specific cofactor in substrate interaction. Furthermore, we provide evidence for the reaction intermediate of PopP2-mediated acetylation, an acetyl-cysteine covalent adduct, lending direct support to the 'ping-pong'-like catalytic mechanism proposed for YopJ effectors. Our study provides critical mechanistic insights into the virulence activity of YopJ class of acetyltransferases.

  1. Alteration of forkhead box O (foxo4 acetylation mediates apoptosis of podocytes in diabetes mellitus.

    Directory of Open Access Journals (Sweden)

    Peter Y Chuang

    Full Text Available The number of kidney podocytes is reduced in diabetic nephropathy. Advanced glycation end products (AGEs accumulate in patients with diabetes and promote the apoptosis of podocyte by activating the forkhead box O4 (Foxo4 transcription factor to increase the expression of a pro-apoptosis gene, Bcl2l11. Using chromatin immunoprecipitation we demonstrate that AGE-modified bovine serum albumin (AGE-BSA enhances Foxo4 binding to a forkhead binding element in the promoter of Bcl2lll. AGE-BSA also increases the acetylation of Foxo4. Lysine acetylation of Foxo4 is required for Foxo4 binding and transcription of Bcl2l11 in podocytes treated with AGE-BSA. The expression of a protein deacetylase that targets Foxo4 for deacetylation, sirtuin (Sirt1, is down regulated in cultured podocytes by AGE-BSA treatment and in glomeruli of diabetic patients. SIRT1 over expression in cultured murine podocytes prevents AGE-induced apoptosis. Glomeruli isolated from diabetic db/db mice have increased acetylation of Foxo4, suppressed expression of Sirt1, and increased expression of Bcl2l11 compared to non-diabetic littermates. Together, our data provide evidence that alteration of Foxo4 acetylation and down regulation of Sirt1 expression in diabetes promote podocyte apoptosis. Strategies to preserve Sirt1 expression or reduce Foxo4 acetylation could be used to prevent podocyte loss in diabetes.

  2. A p300 and SIRT1 Regulated Acetylation Switch of C/EBPα Controls Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    Mohamad A. Zaini

    2018-01-01

    Full Text Available Summary: Cellular metabolism is a tightly controlled process in which the cell adapts fluxes through metabolic pathways in response to changes in nutrient supply. Among the transcription factors that regulate gene expression and thereby cause changes in cellular metabolism is the basic leucine-zipper (bZIP transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα. Protein lysine acetylation is a key post-translational modification (PTM that integrates cellular metabolic cues with other physiological processes. Here, we show that C/EBPα is acetylated by the lysine acetyl transferase (KAT p300 and deacetylated by the lysine deacetylase (KDAC sirtuin1 (SIRT1. SIRT1 is activated in times of energy demand by high levels of nicotinamide adenine dinucleotide (NAD+ and controls mitochondrial biogenesis and function. A hypoacetylated mutant of C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. Our study identifies C/EBPα as a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply. : Zaini et al. show that the transcription factor C/EBPα is acetylated by p300 and deacetylated by the lysine deacetylase SIRT1. Hypoacetylated C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. C/EBPα is a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply. Keywords: C/EBPα, SIRT1, p300, lysine acetylation, mitochondrial function, cellular metabolism, NAD+, gene regulation

  3. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    International Nuclear Information System (INIS)

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo; Neto Paiva, Claudia; Torres Bozza, Marcelo; Rosado Fantappie, Marcelo

    2009-01-01

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1ΔC) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1ΔC were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  4. Acetylation-Dependent Chromatin Reorganization by BRDT, a Testis-Specific Bromodomain-Containing Protein

    Science.gov (United States)

    Pivot-Pajot, Christophe; Caron, Cécile; Govin, Jérôme; Vion, Alexandre; Rousseaux, Sophie; Khochbin, Saadi

    2003-01-01

    The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor. PMID:12861021

  5. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    International Nuclear Information System (INIS)

    Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang

    2014-01-01

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs

  6. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle

    International Nuclear Information System (INIS)

    Cederblad, G.; Carlin, J.I.; Constantin-Teodosiu, D.; Harper, P.; Hultman, E.

    1990-01-01

    Radioisotopic assays for the determination of acetyl-CoA, CoASH, and acetylcarnitine have been modified for application to the amount of human muscle tissue that can be obtained by needle biopsy. In the last step common to all three methods, acetyl-CoA is condensed with [14C]oxaloacetate by citrate synthase to give [14C]-citrate. For determination of CoASH, CoASH is reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to yield acetyl-CoA. In the assay for acetylcarnitine, acetylcarnitine is reacted with CoASH in a reaction catalyzed by carnitine acetyltransferase to form acetyl-CoA. Inclusion of new simple steps in the acetylcarnitine assay and conditions affecting the reliability of all three methods are also described. Acetylcarnitine and free carnitine levels in human rectus abdominis muscle were 3.0 +/- 1.5 (SD) and 13.5 +/- 4.0 mumol/g dry wt, respectively. Values for acetyl-CoA and CoASH were about 500-fold lower, 6.7 +/- 1.8 and 21 +/- 8.9 nmol/g dry wt, respectively. A strong correlation between acetylcarnitine (y) and short-chain acylcarnitine (x), determined as the difference between total and free carnitine, was found in biopsies from the vastus lateralis muscle obtained during intense muscular effort, y = 1.0x + 0.5; r = 0.976

  7. Acetylation curtails nucleosome binding, not stable nucleosome remodeling, by FoxO1

    International Nuclear Information System (INIS)

    Hatta, M.; Liu, F.; Cirillo, L.A.

    2009-01-01

    Transcriptional activity of FoxO factors is controlled through the actions of multiple growth factors signaling through protein kinase B, whereby phosphorylation of FoxO factors inhibits FoxO-mediated transactivation by promoting nuclear export. Phosphorylation of FoxO factors is enhanced by p300-mediated acetylation, which decreases their affinity for DNA. The negative effect of acetylation on FoxO DNA binding, together with nuclear FoxO mobility, is eliminated by over-expression of the de-acetylase Sirt1, suggesting that acetylation mobilizes FoxO factors in chromatin for inducible gene expression. Here, we show that acetylation significantly curtails the affinity of FoxO1 for its binding sites in nucleosomal DNA but has no effect on either stable nucleosome binding or remodeling by this factor. We suggest that, while acetylation provides a first, essential step toward mobilizing FoxO factors for inducible gene repression, additional mechanisms exist for overcoming their inherent capacity to stably bind and remodel nuclear chromatin.

  8. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    Energy Technology Data Exchange (ETDEWEB)

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Neto Paiva, Claudia; Torres Bozza, Marcelo [Departamento de Imunologia, Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Rosado Fantappie, Marcelo, E-mail: fantappie@bioqmed.ufrj.br [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil)

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  9. Patient-Reported Outcome and Observer-Reported Outcome Assessment in Rare Disease Clinical Trials: An ISPOR COA Emerging Good Practices Task Force Report.

    Science.gov (United States)

    Benjamin, Katy; Vernon, Margaret K; Patrick, Donald L; Perfetto, Eleanor; Nestler-Parr, Sandra; Burke, Laurie

    Rare diseases (RDs) affect a small number of people within a population. About 5000 to 8000 distinct RDs have been identified, with an estimated 6% to 8% of people worldwide suffering from an RD. Approximately 75% of RDs affect children. Frequently, these conditions are heterogeneous; many are progressive. Regulatory incentives have increased orphan drug designations and approvals. To develop emerging good practices for RD outcomes research addressing the challenges inherent in identifying, selecting, developing, adapting, and implementing patient-reported outcome (PRO) and observer-reported outcome (ObsRO) assessments for use in RD clinical trials. This report outlines the challenges and potential solutions in determining clinical outcomes for RD trials. It follows the US Food and Drug Administration Roadmap to Patient-Focused Outcome Measurement in Clinical Trials. The Roadmap consists of three columns: 1) Understanding the Disease or Condition, 2) Conceptualizing Treatment Benefit, and 3) Selecting/Developing the Outcome Measure. Challenges in column 1 include factors such as incomplete natural history data and heterogeneity of disease presentation and patient experience. Solutions include using several information sources, for example, clinical experts and patient advocacy groups, to construct the condition's natural history and understand treatment patterns. Challenges in column 2 include understanding and measuring treatment benefit from the patient's perspective, especially given challenges in defining the context of use such as variations in age or disease severity/progression. Solutions include focusing on common symptoms across patient subgroups, identifying short-term outcomes, and using multiple types of COA instruments to measure the same constructs. Challenges in column 3 center around the small patient population and heterogeneity of the condition or study sample. Few disease-specific instruments for RDs exist. Strategies include adapting existing

  10. Preparation of Acetylated Guar Gum – Unsaturated Polyester Composites & Effect of Water on Their Properties

    Directory of Open Access Journals (Sweden)

    David D’Melo

    2012-07-01

    Full Text Available Guar gum has seen extensive use in blends, however, its application as a filler in thermoset composites has as yet not been investigated. The effect of the addition of guar gum and its acetyl derivatives on the kinetics of water diffusion in unsaturated polyester composites was studied. The effect of water on the mechanical properties of the composites was studied with respect to the nature of filler, filler concentration and time of immersion. All the mechanical properties were observed to decrease on exposure to water. Further, it was observed that acetylated guar gum, with a degree of substitution of 0.21, showed the best mechanical properties, surpassing the other filled composites and that of the pure unsaturated polyester. Thus, acetylated guar gum showed promise as eco-friendly filler in composite formulation.

  11. Beta-endorphin and alpha-n-acetyl beta-endorphin; synthesis, conformation and binding parameter

    Energy Technology Data Exchange (ETDEWEB)

    Lovegren, E.S.

    1986-01-01

    Beta-endorphin (EP) is a 31-residue opioid peptide found in many tissues, including the pituitary, brain and reproductive tract. Alpha-amino-acetyl beta-endorphin (AcEP) was characterized spectroscopically by proton nuclear magnetic resonance (NMR) and circular dichroism in deuterated water and trifluoroethanol (TFE). Both EP and AcEP bind to neuroblastoma N2a cells. This binding was not mediated through opiate receptors, and both peptides seemed to bind at common sites. Ovarian immunoreactive-EP levels were determined for immature and mature rates. These levels were found to be responsive to exogenous gonadotropin treatment in immature animals. A large percentage of the immunoreactive-EP is present in follicular fluid, and most of the endorphin-like peptides were acetylated, as measured by radioimmunoassay. Chromatogaphic analysis suggested at least three EP-like species: EP, a carboxy-terminally cleaved and an amino-terminally acetylated EP.

  12. Calix[4]arene-Based Enantioselective Fluorescent Sensors for the Recognition of N-Acetyl-aspartate

    Institute of Scientific and Technical Information of China (English)

    QING Guang-Yan; CHEN Zhi-Hong; WANG Feng; YANG Xi; MENG Ling-Zhi; HE Yong-Bing

    2008-01-01

    Two-armed chiral anion receptors (1 and 2), calix[4]arenes bearing dansyl fluorophore and (1R,2R)- or(1S,2S)-1,2-diphenylethylenediamine binding sites, were prepared and examined for their chiral amino acid anion binding abilities by the fluorescence spectra in dimethylsulfoxide (DMSO). The results of non-linear curve fitting indicate that 1 or 2 forms a 1 : 1 stoichiometry complex with N-acetyl-L-or D-aspartate by multiple hydrogen bonding interactions, exhibiting good enantioselective fluorescent recognition for the enantiomers of N-acetyl-as-partate, [receptor 1: Kass(D)/Kass(L)=6.74; receptor 2: Kass(L)/Kass(D)=6.48]. The clear fluorescent response difference indicates that receptors 1 and 2 could be used as a fluorescent chemosensor for N-Acetyl-aspartate.

  13. Evidence for lysine acetylation in the coat protein of a polerovirus.

    Science.gov (United States)

    Cilia, Michelle; Johnson, Richard; Sweeney, Michelle; DeBlasio, Stacy L; Bruce, James E; MacCoss, Michael J; Gray, Stewart M

    2014-10-01

    Virions of the RPV strain of Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.

  14. Epigenetic regulation of the NR4A orphan nuclear receptor NOR1 by histone acetylation.

    Science.gov (United States)

    Zhao, Yue; Nomiyama, Takashi; Findeisen, Hannes M; Qing, Hua; Aono, Jun; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2014-12-20

    The nuclear receptor NOR1 is an immediate-early response gene implicated in the transcriptional control of proliferation. Since the expression level of NOR1 is rapidly induced through cAMP response element binding (CREB) protein-dependent promoter activation, we investigated the contribution of histone acetylation to this transient induction. We demonstrate that NOR1 transcription is induced by histone deacetylase (HDAC) inhibition and by depletion of HDAC1 and HDAC3. HDAC inhibition activated the NOR1 promoter, increased histone acetylation and augmented the recruitment of phosphorylated CREB to the promoter. Furthermore, HDAC inhibition increased Ser133 phosphorylation of CREB and augmented NOR1 protein stability. These data outline previously unrecognized mechanisms of NOR1 regulation and illustrate a key role for histone acetylation in the rapid induction of NOR1. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Postmortem Toxicology Findings of Acetyl Fentanyl, Fentanyl, and Morphine in Heroin Fatalities in Tampa, Florida

    OpenAIRE

    Pearson, Julia; Poklis, Justin; Poklis, Alphonse; Wolf, Carl; Mainland, Mary; Hair, Laura; Devers, Kelly; Chrostowski, Leszek; Arbefeville, Elise; Merves, Michele

    2015-01-01

    In the last two years, an epidemic of 40 fatal heroin overdose cases has occurred in the Tampa area of Florida. Of these cases, 14 involved fentanyl and acetyl fentanyl. Victim demographics, case histories, toxicology findings, and causes and manners of death for all 40 deaths are presented. In 26 deaths in which acetyl fentanyl or fentanyl were not involved, free and total peripheral blood morphine concentrations were consistent with fatal heroin intoxications, averaging 0.16 mg/L and 0.35 m...

  16. Hydrolysis of wheat B-starch and characterisation of acetylated maltodextrin.

    Science.gov (United States)

    Smrčková, Petra; Horský, Jiří; Šárka, Evžen; Koláček, Jaroslav; Netopilík, Miloš; Walterová, Zuzana; Kruliš, Zdeněk; Synytsya, Andrey; Hrušková, Kateřina

    2013-10-15

    Wheat B-starch was hydrolysed by α-amylase "Liquozyme supra" from Bacillus licheniformis at 90 °C and pH 7. After 2 h, the dextrose equivalent was 18; according to size exclusion chromatography, however, the hydrolysate contained not only dominant malto-oligosaccharides with the degree of polymerisation (DP)40. This non-uniformity of acetylated maltodextrin, both with respect to DP and to DS, must be taken into account in the development of acetylated-maltodextrin applications such as use as plasticisers or compatibilisers in biodegradable composites. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Design of interior-functionalized fully acetylated dendrimers for anticancer drug delivery.

    Science.gov (United States)

    Hu, Jingjing; Su, Yunzhang; Zhang, Hongfeng; Xu, Tongwen; Cheng, Yiyun

    2011-12-01

    In this study, dendrimers was synthesized by introducing functional groups into the interior pockets of fully acetylated dendrimers. NMR techniques including COSY and 2D-NOESY revealed the molecular structures of the synthesized dendrimers and the encapsulation of guest molecule such as methotrexate within their interior pockets. The synthesized polymeric nanocarriers showed much lower cytotoxicity on two cell lines than cationic dendrimers, and exhibited better performance than fully acetylated dendrimers in the sustained release of methotrexate. The results provided a new strategy in the design of non-toxic dendrimers with high performance in the delivery of anti-cancer drugs for clinical applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Micronutrients, N-Acetyl Cysteine, Probiotics and Prebiotics, a Review of Effectiveness in Reducing HIV Progression

    Science.gov (United States)

    Hummelen, Ruben; Hemsworth, Jaimie; Reid, Gregor

    2010-01-01

    Low serum concentrations of micronutrients, intestinal abnormalities, and an inflammatory state have been associated with HIV progression. These may be ameliorated by micronutrients, N-acetyl cysteine, probiotics, and prebiotics. This review aims to integrate the evidence from clinical trials of these interventions on the progression of HIV. Vitamin B, C, E, and folic acid have been shown to delay the progression of HIV. Supplementation with selenium, N-acetyl cysteine, probiotics, and prebiotics has considerable potential, but the evidence needs to be further substantiated. Vitamin A, iron, and zinc have been associated with adverse effects and caution is warranted for their use. PMID:22254046

  19. Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth.

    Science.gov (United States)

    Carabetta, Valerie J; Greco, Todd M; Tanner, Andrew W; Cristea, Ileana M; Dubnau, David

    2016-05-01

    N ε -Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. The past decade highlighted N ε -lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis . To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell

  20. Release behavior and intra-articular biocompatibility of celecoxib-loaded acetyl-capped PCLA-PEG-PCLA thermogels

    NARCIS (Netherlands)

    Petit, Audrey|info:eu-repo/dai/nl/371748461; Sandker, Marjan; Müller, Benno; Meyboom, Ronald; van Midwoud, Paul; Bruin, Peter; Redout, Everaldo M; Versluijs-Helder, Marjan|info:eu-repo/dai/nl/311472699; van der Lest, Chris H A; Buwalda, Sytze J|info:eu-repo/dai/nl/339146850; de Leede, Leo G J; Vermonden, Tina|info:eu-repo/dai/nl/275124517; Kok, Robbert Jan|info:eu-repo/dai/nl/170678326; Weinans, Harrie; Hennink, Wim E|info:eu-repo/dai/nl/070880409

    In this study, we investigated the in vitro and in vivo properties and performance of a celecoxib-loaded hydrogel based on a fully acetyl-capped PCLA-PEG-PCLA triblock copolymer. Blends of different compositions of celocoxib, a drug used for pain management in osteoarthritis, and the acetyl-capped

  1. A proteome-scale study on in vivo protein N(α)-acetylation using an optimized method

    DEFF Research Database (Denmark)

    Zhang, Xumin; Engholm-Keller, Kasper; Højrup, Peter

    2011-01-01

    Protein N-terminal acetylation (N(α)-acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α)-modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be ...

  2. Structural studies on 4-O-acetyl-α-N-acetylneuraminyl-(2→3)-lactose, the main oligosaccharide in echidna milk

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Kamerling, J.P.; Dorland, L.; Halbeek, H. van; Messer, M.; Schauer, R.

    1982-01-01

    The main oligosaccharide (50%) in the milk of the Australian echidna (Tachyglossus aculeatus) has been identified unequivocally as 4-O-acetyl-α-N-acetylneur-aminyl-(2→3)-lactose. The 4-O-acetyl substituent of the sialic acid residue was characterised by g.l.c.-m.s. of the isolated (after mild, acid

  3. The bacterial two-hybrid system uncovers the involvement of acetylation in regulating of Lrp activity in Salmonella Typhimurium

    Directory of Open Access Journals (Sweden)

    Ran Qin

    2016-11-01

    Full Text Available Nε-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat - or deacetylase CobB-mediated acetylation. Then, the in vitro (deacetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36 in helix-turn-helix (HTH DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.

  4. Inhibition of Different Histone Acetyltransferases (HATs) Uncovers Transcription-Dependent and -Independent Acetylation-Mediated Mechanisms in Memory Formation

    Science.gov (United States)

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-01-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied…

  5. The Global Acetylome of the Human Pathogen Vibrio cholerae V52 Reveals Lysine Acetylation of Major Transcriptional Regulators

    DEFF Research Database (Denmark)

    Jers, Carsten; Ravikumar, Vaishnavi; Lezyk, Mateusz Jakub

    2018-01-01

    Protein lysine acetylation is recognized as an important reversible post translational modification in all domains of life. While its primary roles appear to reside in metabolic processes, lysine acetylation has also been implicated in regulating pathogenesis in bacteria. Several global lysine...... acetylome analyses have been carried out in various bacteria, but thus far there have been no reports of lysine acetylation taking place in the important human pathogen Vibrio cholerae. In this study, we analyzed the lysine acetylproteome of the human pathogen V. cholerae V52. By applying a combination...... in direct regulation of virulence in V. cholerae were acetylated. In conclusion, this is the first global protein lysine acetylome analysis of V. cholerae and should constitute a valuable resource for in-depth studies of the impact of lysine acetylation in pathogenesis and other cellular processes....

  6. Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

    Directory of Open Access Journals (Sweden)

    L. López-Contreras

    2013-01-01

    Full Text Available Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  7. Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Wagner, Sebastian A; Horn, Heiko

    2011-01-01

    Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines....... With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites...... between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines...

  8. Arabidopsis NATA1 Acetylates Putrescine and Decreases Defense-Related Hydrogen Peroxide Accumulation1[OPEN

    Science.gov (United States)

    Preuss, Aileen S.

    2016-01-01

    Biosynthesis of the polyamines putrescine, spermidine, and spermine is induced in response to pathogen infection of plants. Putrescine, which is produced from Arg, serves as a metabolic precursor for longer polyamines, including spermidine and spermine. Polyamine acetylation, which has important regulatory functions in mammalian cells, has been observed in several plant species. Here we show that Arabidopsis (Arabidopsis thaliana) N-ACETYLTRANSFERASE ACTIVITY1 (NATA1) catalyzes acetylation of putrescine to N-acetylputrescine and thereby competes with spermidine synthase for a common substrate. NATA1 expression is strongly induced by the plant defense signaling molecule jasmonic acid and coronatine, an effector molecule produced by DC3000, a Pseudomonas syringae strain that initiates a virulent infection in Arabidopsis ecotype Columbia-0. DC3000 growth is reduced in nata1 mutant Arabidopsis, suggesting a role for NATA1-mediated putrescine acetylation in suppressing antimicrobial defenses. During infection by P. syringae and other plant pathogens, polyamine oxidases use spermidine and spermine as substrates for the production of defense-related H2O2. Compared to wild-type Columbia-0 Arabidopsis, the response of nata1mutants to P. syringae infection includes reduced accumulation of acetylputrescine, greater abundance of nonacetylated polyamines, elevated H2O2 production by polyamine oxidases, and higher expression of genes related to pathogen defense. Together, these results are consistent with a model whereby P. syringae growth is improved in a targeted manner through coronatine-induced putrescine acetylation by NATA1. PMID:27208290

  9. Oxidative stress-triggered interactions between the succinyl- and acetyl-proteomes of rice leaves.

    Science.gov (United States)

    Zhou, Heng; Finkemeier, Iris; Guan, Wenxue; Tossounian, Maria-Armineh; Wei, Bo; Young, David; Huang, Jingjing; Messens, Joris; Yang, Xibin; Zhu, Jun; Wilson, Michael H; Shen, Wenbiao; Xie, Yanjie; Foyer, Christine H

    2018-05-01

    Protein lysine acylations, such as succinylation and acetylation, are important post-translational modification (PTM) mechanisms, with key roles in cellular regulation. Antibody-based affinity enrichment, high-resolution liquid chromatography mass spectrometry analysis, and integrated bioinformatics analysis were used to characterize the lysine succinylome (K suc ) and acetylome (K ace ) of rice leaves. In total, 2,593 succinylated and 1,024 acetylated proteins were identified, of which 723 were simultaneously acetylated and succinylated. Proteins involved in photosynthetic carbon metabolism such as the large and small subunits of RuBisCO, ribosomal functions, and other key processes were subject to both PTMs. Preliminary insights into oxidant-induced changes to the rice acetylome and succinylome were gained from treatments with hydrogen peroxide. Exposure to oxidative stress did not regulate global changes in the rice acetylome or succinylome but rather led to modifications on a specific subset of the identified sites. De-succinylation of recombinant catalase (CATA) and glutathione S-transferase (OsGSTU6) altered the activities of these enzymes showing that this PTM may have a regulatory function. These findings not only greatly extend the list of acetylated and/or succinylated proteins but they also demonstrate the close cooperation between these PTMs in leaf proteins with key metabolic functions. © 2017 John Wiley & Sons Ltd.

  10. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate

    2012-01-01

    ,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals...

  11. CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

    Czech Academy of Sciences Publication Activity Database

    Dušková, Eva; Hnilicová, Jarmila; Staněk, David

    2014-01-01

    Roč. 11, č. 7 (2014), s. 865-874 ISSN 1547-6286 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68378050 Keywords : alternative splicing * fibronectin * p300 * histone acetylation * promoter Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.974, year: 2014

  12. Properties of retrograded and acetylated starch produced via starch extrusion or starch hydrolysis with pullulanase.

    Science.gov (United States)

    Kapelko, M; Zięba, T; Gryszkin, A; Styczyńska, M; Wilczak, A

    2013-09-12

    The aim of the present study was to determine the impact of serial modifications of starch, including firstly starch extrusion or hydrolysis with pullulanase, followed by retrogradation (through freezing and defrosting of pastes) and acetylation (under industrial conditions), on its susceptibility to amylolysis. The method of production had a significant effect on properties of the resultant preparations, whilst the direction and extent of changes depended on the type of modification applied. In the produced starch esters, the degree of substitution, expressed by the per cent of acetylation, ranged from 3.1 to 4.4 g/100 g. The acetylation had a significant impact on contents of elements determined with the atomic emission spectrometry, as it contributed to an increased Na content and decreased contents of Ca and K. The DSC thermal characteristics enabled concluding that the modifications caused an increase in temperatures and a decrease in heat of transition (or its lack). The acetylation of retrograded starch preparations increased their solubility in water and water absorbability. The modifications were found to exert various effects on the rheological properties of pastes determined based on the Brabender's pasting characteristics and flow curves determined with the use of an oscillatory-rotating viscosimeter. All starch acetates produced were characterized by ca. 40% resistance to amylolysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Kinetics of Acid Hydrolysis of Water-Soluble Spruce O-Acetyl Galactoglucomannans

    NARCIS (Netherlands)

    Xu, C.; Pranovich, A.; Vahasalo, L.; Hemming, J.; Holmbom, B.; Schols, H.A.; Willfor, S.

    2008-01-01

    Water-soluble O-acetyl galactoglucomannan (GGM) is a softwood-derived polysaccharide, which can be extracted on an industrial scale from wood or mechanical pulping waters and now is available in kilogram scale for research and development of value-added products. To develop applications of GGM,

  14. [PHI regulates histone methylation and acetylation in Burkitt lymphoma Daudi cell line].

    Science.gov (United States)

    Hong, Ling-Ling; Ma, Xu-Dong; Huang, Yi-Qun

    2011-02-01

    This study was purposed to investigate the effects of phenylhexyl isothiocyanate (PHI) on Burkitt lymphoma Daudi cell line and regulation of histone acetylation and methylation in Daudi cells, and to explore the potential mechanism. The apoptotic rate of Daudi cells treated with PHI was measured by flow cytometry, the changes of histone H3 and H4 acetylation, histone H3K9 and H3K4 methylation in Daudi cells treated with PHI were detected by Western blot. The results showed that PHI could induce apoptosis of Daudi cells, increased the acetylation level of H3 and H4, enhanced the methylation of H3K4, but reduced the methylation of H3K9. It is concluded that the PHI can up-regulate the acetylation level of histone H3 associated with transcription stimulation and the methylation of histone H3K4, down-regulate the methylation on histone H3K9 associated with transcription inhibition, promotes the apoptosis of Daudi cells. PHI may be a potential agent for target therapy of lymphoma.

  15. Hydrolysis of wheat B-starch and characterisation of acetylated maltodextrin

    Czech Academy of Sciences Publication Activity Database

    Smrčková, P.; Horský, Jiří; Šárka, E.; Koláček, J.; Netopilík, Miloš; Walterová, Zuzana; Kruliš, Zdeněk; Synytsya, A.; Hrušková, K.

    2013-01-01

    Roč. 98, č. 1 (2013), s. 43-49 ISSN 0144-8617 R&D Projects: GA ČR GA525/09/0607 Institutional research plan: CEZ:AV0Z40500505 Keywords : wheat B-starch * α-amylase * acetylated maltodextrin Subject RIV: JI - Composite Materials Impact factor: 3.916, year: 2013

  16. Acetylation regulates WRN catalytic activities and affects base excision DNA repair

    DEFF Research Database (Denmark)

    Muftuoglu, Meltem; Kusumoto, Rika; Speina, Elzbieta

    2008-01-01

    The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone...... acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription....

  17. Indirect flow injection determination of N-acetyl-L-cysteine using cerium(IV) and ferroin

    International Nuclear Information System (INIS)

    Vieira, Heberth Juliano; Fatibello-Filho, Orlando

    2005-01-01

    An indirect flow injection spectrophotometric procedure is proposed for the determination of N-acetyl-L-cysteine in pharmaceutical formulations. In this system, ferroin ([Fe(II)-(fen) 2 ] 2+ ) in excess, with a strong absorption at 500 nm, is oxidized by cerium(IV) yielding cerium(III) and [Fe(III)-(fen) 2 ] 3+ (colorless), thus producing a baseline. When N-acetyl-L-cysteine solution is introduced into the flow injection system, it reacts with cerium(IV) increasing the analytical signal in proportion to the drug concentration. Under optimal experimental conditions, the linearity of the analytical curve for N-acetyl-L-cysteine ranged from 6.5x10 -6 to 1.3x10 -4 mol L -1 . The detection limit was 5.0x10 -6 mol L -1 and recoveries between 98.0 and 106% were obtained. The sampling frequency was 60 determinations per hour and the RSD was smaller than 1.4% for 2.2x10 -5 mol L -1 N-acetyl-L-cysteine. (author)

  18. Urinary excretion of N-acetyl-S-allyl-L-cystein upon garlic consumption by human volunteers.

    NARCIS (Netherlands)

    de Rooij, B.M.; Boogaard, P.J.; Rijksen, D.A.; Commandeur, J.N.M.; Vermeulen, N.P.E.

    1996-01-01

    N-Acetyl-S-allyl-L-cysteine (allylmercapturic acid, ALMA) was previously detected in urine from humans consuming garlic. Exposure of rats to allyl halides is also known to lead to excretion of ALMA in urine. ALMA is a potential biomarker for exposure assessment of workers exposed to allyl halides.

  19. Micronutrients, N-Acetyl Cysteine, Probiotics and Prebiotics, A Review of Effectiveness in Reducing HIV Progression

    NARCIS (Netherlands)

    R.B.S. Hummelen (Ruben); J. Hemsworth (Jaimie); G.K. Reid (Gregor)

    2010-01-01

    textabstractLow serum concentrations of micronutrients, intestinal abnormalities, and an inflammatory state have been associated with HIV progression. These may be ameliorated by micronutrients, N-acetyl cysteine, probiotics, and prebiotics. This review aims to integrate the evidence from clinical

  20. A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3

    NARCIS (Netherlands)

    Remoroza, C.A.; Wagenknecht, M.; Buchholt, H.C.; Moerschbacher, B.M.; Schols, H.A.; Gruppen, H.

    2014-01-01

    A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional

  1. Direct acetylation of sunflower oil in the presence of boron trioxide ...

    African Journals Online (AJOL)

    Lubrication properties of sunflower oil have been modified by epoxidation in the first step and acetylation of the obtained epoxide in the second step. Epoxidation has been followed in dichloromethane solution in the presence of hydrogen peroxide and acetic acid as oxidizing agent and sulfuric acid as catalyst. The reaction ...

  2. Arabinose content of arabinoxylans contributes to flexibility of acetylated arabinoxylan films

    NARCIS (Netherlands)

    Stepan, A.M.; Hoïje, A.; Schols, H.A.; Waard, de P.; Gatenholm, P.

    2012-01-01

    Arabinoxylans (AX) from rye were partly debranched by chemical hydrolysis methods, and AXs differing in arabinosyl substitution were acetylated using chemical methods. The resulting materials are film forming, and these films underwent molecular structural analysis and were tested for their material

  3. Hydrolysis of Wheat Arabinoxylan by Two Acetyl Xylan Esterases from Chaetomium thermophilum

    DEFF Research Database (Denmark)

    Tong, Xiaoxue; Lange, Lene; Grell, Morten Nedergaard

    2015-01-01

    The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 3.1.1.72) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has...

  4. Green starch conversions : Studies on starch acetylation in densified CO2

    NARCIS (Netherlands)

    Muljana, Henky; Picchioni, Francesco; Heeres, Hero J.; Janssen, Leon P. B. M.

    2010-01-01

    The acetylation of potato starch with acetic anhydride (AAH) and sodium acetate (NaOAc) as catalyst in densified CO2 was explored in a batch reactor setup. The effects of process variables such as pressure (6-9.8 MPa), temperature (40-90 degrees C), AAH to starch ratio (2-5 mol/mol AGU), NaOAc to

  5. Effect of the acetylation process on native starches of yam (Dioscorea spp.

    Directory of Open Access Journals (Sweden)

    Jairo Salcedo Mendoza

    2016-07-01

    Full Text Available In Colombia, it is necessary to produce native and modified starches for the use of amylaceous raw materials of major socioeconomic importance. In this study, the effects of the acetylation process on structural, morphological and functional properties of native starches yam, Dioscorea spp. (D. alata and D. rotundata were evaluated. Chemical modification by esterification with acetic anhydride was performed at different reaction times, and morphological and structural changes were assessed using the following techniques: infrared spectroscopy (FTIR, X-ray diffraction and scanning electron microscopy (SEM. Acetylation produced slight changes in the granule morphology, and a decreased degree of crystallinity (DC associated with a slight increase in the amylose content was observed. The introduction of acetyl groups into the starch structure caused a decrease in the gelatinization temperature and an increased retro gradation tendency. The acetylated starches had low degrees of substitution (DS<0.2, meaning they can be used in the food industry, considering that they showed greater stability, greater water absorption capacity and better solubility than native starches.

  6. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Schölz, Christian; Wagner, Sebastian A

    2013-01-01

    Recent studies have shown that lysines can be posttranslationally modified by various types of acylations. However, except for acetylation, very little is known about their scope and cellular distribution. We mapped thousands of succinylation sites in bacteria (E. coli), yeast (S. cerevisiae), hu...

  7. Acetyl salicylic acid–ZnAl layered double hydroxide functional nanohybrid for skin care application

    CSIR Research Space (South Africa)

    Mosangi, Damodar

    2016-10-01

    Full Text Available In this study, a pharmaceutically active ingredient, acetyl salicylic acid (ASA), was intercalated into ZnAl layered double hydroxide (LDH). The LDH–ASA nanohybrid material was characterized by XRD, FTIR, SEM, ICP-MS, TEM and TGA. Successful...

  8. Micronutrients, N-acetyl cysteine, probiotics and prebiotics, a review of effectiveness in reducing HIV progression

    NARCIS (Netherlands)

    R.B.S. Hummelen (Ruben); J. Hemsworth (Jaimie); G. Reid (Gregor)

    2010-01-01

    textabstractLow serum concentrations of micronutrients, intestinal abnormalities, and an inflammatory state have been associated with HIV progression. These may be ameliorated by micronutrients, N-acetyl cysteine, probiotics, and prebiotics. This review aims to integrate the evidence from clinical

  9. Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

    NARCIS (Netherlands)

    Levisson, M.; Han, G.W.; Deller, M.C.; Hendriks, S.N.A.; Oost, van der J.; Kengen, S.W.M.

    2012-01-01

    TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity

  10. Specific acid catalyzed deuteration of the acetyl groups of 2,4-diacetyldeuterohemin-OMe

    International Nuclear Information System (INIS)

    Oster, O.; Neireiter, G.W.; Gurd, F.R.N.

    1975-01-01

    The methyl group of the acetyl groups in 2,4-diacetyldeuterohemin-OMe has been selectively deuterated. After removal of the iron, D 6 -2,4-diacetyl-deuteroporphyrin-OMe can be reduced to the corresponding hematoporphyrin and subsequent dehydration gives deuterated vinylic groups for protoporphyrin IX-OMe. (orig.) [de

  11. Ficolins and FIBCD1: Soluble and membrane bound pattern recognition molecules with acetyl group selectivity

    DEFF Research Database (Denmark)

    Thomsen, Theresa; Schlosser, Anders; Holmskov, Uffe

    2011-01-01

    as pattern recognition molecules. Ficolins are soluble oligomeric proteins composed of trimeric collagen-like regions linked to fibrinogen-related domains (FReDs) that have the ability to sense molecular patterns on both pathogens and apoptotic cell surfaces and activate the complement system. The ficolins......D-containing molecules, and discusses structural resemblance but also diversity in recognition of acetylated ligands....

  12. Correlation of Global N-Acetyl Aspartate With Cognitive Impairment in Multiple Sclerosis

    DEFF Research Database (Denmark)

    Kahr Mathiesen, Henrik; Jonsson, Agnete; Tscherning, Thomas

    2006-01-01

    BACKGROUND: Whole-brain N-acetyl aspartate (NAA), a measure of neuronal function, can be assessed by multislice echo-planar spectroscopic imaging. OBJECTIVE: To test the hypothesis that the global brain NAA/creatine (Cr) ratio is a better predictor of cognitive dysfunction in multiple sclerosis...

  13. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    International Nuclear Information System (INIS)

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

    2011-01-01

    Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  14. Acetylation Is Crucial for p53-Mediated Ferroptosis and Tumor Suppression

    Directory of Open Access Journals (Sweden)

    Shang-Jui Wang

    2016-10-01

    Full Text Available Although previous studies indicate that loss of p53-mediated cell cycle arrest, apoptosis, and senescence does not completely abrogate its tumor suppression function, it is unclear how the remaining activities of p53 are regulated. Here, we have identified an acetylation site at lysine K98 in mouse p53 (or K101 for human p53. Whereas the loss of K98 acetylation (p53K98R alone has very modest effects on p53-mediated transactivation, simultaneous mutations at all four acetylation sites (p534KR: K98R+ 3KR[K117R+K161R+K162R] completely abolish its ability to regulate metabolic targets, such as TIGAR and SLC7A11. Notably, in contrast to p533KR, p534KR is severely defective in suppressing tumor growth in mouse xenograft models. Moreover, p534KR is still capable of inducing the p53-Mdm2 feedback loop, but p53-dependent ferroptotic responses are markedly abrogated. Together, these data indicate the critical role of p53 acetylation in ferroptotic responses and its remaining tumor suppression activity.

  15. Effects of N-acetyl cysteine on lipid levels and on leukocyte and ...

    African Journals Online (AJOL)

    Introduction: Many of studies have shown that increased lipid levels play a significant role in the pathogenesis of atherosclerosis after splenectomy. We investigated the effects of N-acetyl cysteine (NAC) on lipid parameters and leukocyte and platelet (PLT) levels following splenectomy. Materials and Methods: 32 Sprague.

  16. Termite and fungal resistance of in situ polymerized tributyltin acrylate and acetylated Indonesian and USA wood

    Science.gov (United States)

    Rebecca E. Ibach; Yusuf Sudo Hadi; Dodi Nandika; Sulaeman Yusuf; Yuliati Indrayani

    2000-01-01

    Wood [Indonesian pine (IP), Indonesian Jabon (IJ) and USA southern yellow pine (USP)] was either in situ polymerized with tributyltin acrylate (TBTA) or acetylated and then exposed to termite and fungal degradation both in laboratory tests and field exposure. The TBTA woods had an average weight percent gain (WPG) of 11% for IP, 12% for IJ, and 10% for USP. The...

  17. The effect of N-acetyl-L-cysteine on the viscosity of ileal neobladder mucus.

    NARCIS (Netherlands)

    Schrier, B.P.; Lichtendonk, W.J.; Witjes, J.A.

    2002-01-01

    N-acetyl-L-cysteine (NAC) proved to be an effective mucolytic in pulmonary secretions. Our goal was to investigate the in vitro effect of NAC on viscosity of ileal neobladder mucus. The urine of a patient with an ileal neobladder was collected during the first 7 days postoperatively and stored in a

  18. Characterisation of a novel homodimeric N-acetyl-β-D-glucosaminidase from Streptococcus gordonii

    International Nuclear Information System (INIS)

    Harty, Derek W.S.; Chen Yingjian; Simpson, Christine L.; Berg, Tracey; Cook, Simon L.; Mayo, John A.; Hunter, Neil; Jacques, Nicholas A.

    2004-01-01

    An N-acetyl-β-D-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS-PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-β-D-glucosaminide, 4MU-N-acetyl-β-D-galactosaminide, 4-MU-β-D-N,N ' -diacetylchitobioside, and 4-MU-β-D-N,N ' ,N''-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 deg. C. The K m for 4-MU-β-D-N,N ' ,N''-chitotrioside, 45 μM, was the lowest for all the substrates tested. Hg 2+ , Cu 2+ , Fe 2+ , and Zn 2+ completely inhibited while Co 2+ , Mn 2+ , and Ni 2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-β-D-glucosaminidase activities

  19. Lifespan extension and increased resistance to environmental stressors by N-Acetyl-L-Cysteine in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Seung-Il Oh

    2015-05-01

    Full Text Available OBJECTIVE: This study was performed to determine the effect of N-acetyl-L-cysteine, a modified sulfur-containing amino acid that acts as a strong cellular antioxidant, on the response to environmental stressors and on aging in C. elegans. METHOD: The survival of worms under oxidative stress conditions induced by paraquat was evaluated with and without in vivo N-acetyl-L-cysteine treatment. The effect of N-acetyl-L-cysteine on the response to other environmental stressors, including heat stress and ultraviolet irradiation (UV, was also monitored. To investigate the effect on aging, we examined changes in lifespan, fertility, and expression of age-related biomarkers in C. elegans after N-acetyl-L-cysteine treatment. RESULTS: Dietary N-acetyl-L-cysteine supplementation significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans. In addition, N-acetyl-L-cysteine supplementation significantly extended both the mean and maximum lifespan of C. elegans. The mean lifespan was extended by up to 30.5% with 5 mM N-acetyl-L-cysteine treatment, and the maximum lifespan was increased by 8 days. N-acetyl-L-cysteine supplementation also increased the total number of progeny produced and extended the gravid period of C. elegans. The green fluorescent protein reporter assay revealed that expression of the stress-responsive genes, sod-3 and hsp-16.2, increased significantly following N-acetyl-L-cysteine treatment. CONCLUSION: N-acetyl-L-cysteine supplementation confers a longevity phenotype in C. elegans, possibly through increased resistance to environmental stressors.

  20. Regulation of Nur77 protein turnover through acetylation and deacetylation induced by p300 and HDAC1.

    Science.gov (United States)

    Kang, Shin-Ae; Na, Hyelin; Kang, Hyun-Jin; Kim, Sung-Hye; Lee, Min-Ho; Lee, Mi-Ock

    2010-09-15

    Although the roles of Nur77, an orphan member of the nuclear hormone receptor superfamily, in the control of cellular proliferation, apoptosis, inflammation, and glucose metabolism, are well recognized, the molecular mechanism regulating the activity and expression of Nur77 is not fully understood. Acetylation of transcription factors has emerged recently as a major post-translational modification that regulates protein stability and transcriptional activity. Here, we examined whether Nur77 is acetylated, and we characterized potential associated factors. First, Nur77 was found to be an acetylated protein when examined by immunoprecipitation and western blotting using acetyl protein-specific antibodies. Second, expression of p300, which possesses histone acetyltransferase activity, enhanced the acetylation and protein stability of Nur77. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, also increased Nur77 acetylation. Among the several types of HDACs, HDAC1 was found as the major enzyme affecting protein level of Nur77. HDAC1 decreased the acetylation level, protein level, and transcriptional activity of Nur77. Interestingly, overexpression of Nur77 induced expression of both p300 and HDAC1. Finally, the expression of Nur77 increased along with that of p300, but decreased with induction of HDAC1 after treatment with epithelial growth factor, nerve growth factor, or 6-mercaptopurine, suggesting that the self-control of the acetylation status contributes to the transient induction of Nur77 protein. Taken together, these results demonstrate that acetylation of Nur77 is modulated by p300 and HDAC1, and suggest that acetylation is an important post-translational modification for the rapid turnover of Nur77 protein. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Rapid Quantification of Low-Viscosity Acetyl-Triacylglycerols Using Electrospray Ionization Mass Spectrometry.

    Science.gov (United States)

    Bansal, Sunil; Durrett, Timothy P

    2016-09-01

    Acetyl-triacylglycerols (acetyl-TAG) possess an sn-3 acetate group, which confers useful chemical and physical properties to these unusual triacylglycerols (TAG). Current methods for quantification of acetyl-TAG are time consuming and do not provide any information on the molecular species profile. Electrospray ionization mass spectrometry (ESI-MS)-based methods can overcome these drawbacks. However, the ESI-MS signal intensity for TAG depends on the aliphatic chain length and unsaturation index of the molecule. Therefore response factors for different molecular species need to be determined before any quantification. The effects of the chain length and the number of double-bonds of the sn-1/2 acyl groups on the signal intensity for the neutral loss of short chain length sn-3 groups were quantified using a series of synthesized sn-3 specific structured TAG. The signal intensity for the neutral loss of the sn-3 acyl group was found to negatively correlated with the aliphatic chain length and unsaturation index of the sn-1/2 acyl groups. The signal intensity of the neutral loss of the sn-3 acyl group was also negatively correlated with the size of that chain. Further, the position of the group undergoing neutral loss was also important, with the signal from an sn-2 acyl group much lower than that from one located at sn-3. Response factors obtained from these analyses were used to develop a method for the absolute quantification of acetyl-TAG. The increased sensitivity of this ESI-MS-based approach allowed successful quantification of acetyl-TAG in various biological settings, including the products of in vitro enzyme activity assays.

  2. Serum Acetyl Cholinesterase as a Biomarker of Arsenic Induced Neurotoxicity in Sprague-Dawley Rats

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2005-04-01

    Full Text Available Arsenic is an environmental toxicant, and one of the major mechanisms by which it exerts its toxic effect is through an impairment of cellular respiration by inhibition of various mitochondrial enzymes, and the uncoupling of oxidative phosphorylation. Most toxicity of arsenic results from its ability to interact with sulfhydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Most toxicity of arsenic results from its ability to interact with sulfhydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Recent studies have pointed out that arsenic toxicity is associated with the formation of reactive oxygen species, which may cause severe injury/damage to the nervous system. The main objective of this study was to conduct biochemical analysis to determine the effect of arsenic trioxide on the activity of acetyl cholinesterase; a critical important nervous system enzyme that hydrolyzes the neurotransmitter acetylcholine. Four groups of six male rats each weighing an average 60 + 2 g were used in this study. Arsenic trioxide was intraperitoneally administered to the rats at the doses of 5, 10, 15, 20mg/kg body weight (BW, one dose per 24 hour given for five days. A control group was also made of 6 animals injected with distilled water without chemical. Following anaesthesia, blood specimens were immediately collected using heparinized syringes, and acetyl cholinesterase detection and quantification were performed in serum samples by spectrophotometry. Arsenic trioxide exposure significantly decreased the activity of cholinesterase in the Sprague-Dawley rats. Acetyl cholinesterase activities of 6895 + 822, 5697 + 468, 5069 + 624, 4054 + 980, and 3158 + 648 U/L were recorded for 0, 5, 10, 15, and 20 mg/kg, respectively; indicating a gradual decrease in acetyl cholinesterase activity with increasing doses of arsenic. These findings indicate that acetyl

  3. Hippocampal histone acetylation regulates object recognition and the estradiol-induced enhancement of object recognition.

    Science.gov (United States)

    Zhao, Zaorui; Fan, Lu; Fortress, Ashley M; Boulware, Marissa I; Frick, Karyn M

    2012-02-15

    Histone acetylation has recently been implicated in learning and memory processes, yet necessity of histone acetylation for such processes has not been demonstrated using pharmacological inhibitors of histone acetyltransferases (HATs). As such, the present study tested whether garcinol, a potent HAT inhibitor in vitro, could impair hippocampal memory consolidation and block the memory-enhancing effects of the modulatory hormone 17β-estradiol E2. We first showed that bilateral infusion of garcinol (0.1, 1, or 10 μg/side) into the dorsal hippocampus (DH) immediately after training impaired object recognition memory consolidation in ovariectomized female mice. A behaviorally effective dose of garcinol (10 μg/side) also significantly decreased DH HAT activity. We next examined whether DH infusion of a behaviorally subeffective dose of garcinol (1 ng/side) could block the effects of DH E2 infusion on object recognition and epigenetic processes. Immediately after training, ovariectomized female mice received bilateral DH infusions of vehicle, E2 (5 μg/side), garcinol (1 ng/side), or E2 plus garcinol. Forty-eight hours later, garcinol blocked the memory-enhancing effects of E2. Garcinol also reversed the E2-induced increase in DH histone H3 acetylation, HAT activity, and levels of the de novo methyltransferase DNMT3B, as well as the E2-induced decrease in levels of the memory repressor protein histone deacetylase 2. Collectively, these findings suggest that histone acetylation is critical for object recognition memory consolidation and the beneficial effects of E2 on object recognition. Importantly, this work demonstrates that the role of histone acetylation in memory processes can be studied using a HAT inhibitor.

  4. Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets.

    Science.gov (United States)

    Marimuthu, Srinivasan; Chivukula, Raghavender S V; Alfonso, Lloyd F; Moridani, Majid; Hagen, Fred K; Bhat, G Jayarama

    2011-11-01

    Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immuno-affinity purification, mass spectrometry and immuno-blotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeleton proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiment showed that aspirin also acetylated glucose-6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6 phosphate dehydrogenase activity. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

  5. Missing value imputation for microarray gene expression data using histone acetylation information

    Directory of Open Access Journals (Sweden)

    Feng Jihua

    2008-05-01

    Full Text Available Abstract Background It is an important pre-processing step to accurately estimate missing values in microarray data, because complete datasets are required in numerous expression profile analysis in bioinformatics. Although several methods have been suggested, their performances are not satisfactory for datasets with high missing percentages. Results The paper explores the feasibility of doing missing value imputation with the help of gene regulatory mechanism. An imputation framework called histone acetylation information aided imputation method (HAIimpute method is presented. It incorporates the histone acetylation information into the conventional KNN(k-nearest neighbor and LLS(local least square imputation algorithms for final prediction of the missing values. The experimental results indicated that the use of acetylation information can provide significant improvements in microarray imputation accuracy. The HAIimpute methods consistently improve the widely used methods such as KNN and LLS in terms of normalized root mean squared error (NRMSE. Meanwhile, the genes imputed by HAIimpute methods are more correlated with the original complete genes in terms of Pearson correlation coefficients. Furthermore, the proposed methods also outperform GOimpute, which is one of the existing related methods that use the functional similarity as the external information. Conclusion We demonstrated that the using of histone acetylation information could greatly improve the performance of the imputation especially at high missing percentages. This idea can be generalized to various imputation methods to facilitate the performance. Moreover, with more knowledge accumulated on gene regulatory mechanism in addition to histone acetylation, the performance of our approach can be further improved and verified.

  6. 17ß-Estradiol Regulates Histone Alterations Associated with Memory Consolidation and Increases "Bdnf" Promoter Acetylation in Middle-Aged Female Mice

    Science.gov (United States)

    Fortress, Ashley M.; Kim, Jaekyoon; Poole, Rachel L.; Gould, Thomas J.; Frick, Karyn M.

    2014-01-01

    Histone acetylation is essential for hippocampal memory formation in young adult rodents. Although dysfunctional histone acetylation has been associated with age-related memory decline in male rodents, little is known about whether histone acetylation is altered by aging in female rodents. In young female mice, the ability of 17ß-estradiol…

  7. Accurate quantification of site-specific acetylation stoichiometry reveals the impact of sirtuin deacetylase CobB on the E. coli acetylome

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Satpathy, Shankha; Hansen, Bogi Karbech

    2017-01-01

    B suppressed acetylation to lower than median stoichiometry in WT, ptaΔ, and ackAΔ cells. Together, our results provide a detailed view of acetylation stoichiometry in E. coli and suggest an evolutionarily conserved function of Sirtuin deacetylases in suppressing low stoichiometry acetylation....

  8. An unusual insertion/deletion in the gene encoding the β-subunit of propionyl-CoA carboxylase is a frequent mutation in Caucasian propionic acidemia

    International Nuclear Information System (INIS)

    Tahara, T.; Kraus, J.P.; Rosenberg, L.E.

    1990-01-01

    Propionic acidemia is an inherited disorder of organic acid metabolism that is caused by deficiency of propionly-CoA carboxylase. Affected patients fall into two complementation groups, pccA and pccBC (subgroups B, C, and BC), resulting from deficiency of the nonidentical α and β subunits of PCC, respectively. The authors have detected an unusual insertion/deletion in the DNA of patients from the pccBC and pccC subgroups that replaces 14 nucleotides in the coding sequence of the β subunit with 12 nucleotides unrelated to this region of the gene. Among 14 unrelated Caucasian patients in the pccBc complementation group, this unique mutation was found in 8 of 28 mutant alleles examined. Mutant allele-specific oligonucleotide hybridization to amplified genomic DNAs revealed that the inserted 12 nucleotides do not originate in an ∼1000-bp region around the mutation. In the course of the investigation, they identified another mutation in the same exon: a 3-bp in-frame deletion that eliminates one of two isoleucine codons immediately preceding the Msp I site. Two unrelated patients were compound heterozygotes for this single-codon deletion and for the insertion/deletion described above. They conclude that either there is a propensity for the PCC β-subunit gene to undergo mutations of this sort at this position or, more likely, the mutations in all of the involved Caucasian patients have a common origin in preceding generations

  9. Silencing ribulose-1,5-bisphosphate carboxylase/oxygenase expression does not disrupt nitrogen allocation to defense after simulated herbivory in Nicotiana attenuata.

    Science.gov (United States)

    Stanton, Mariana A; Ullmann-Zeunert, Lynn; Wielsch, Natalie; Bartram, Stefan; Svatoš, Aleš; Baldwin, Ian T; Groten, Karin

    2013-01-01

    Ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) is the most abundant protein on the planet and in addition to its central role in photosynthesis it is thought to function as a nitrogen (N)-storage protein and a potential source of N for defense biosynthesis in plants. In a recent study in the wild tobacco Nicotiana attenuata, we showed that the decrease in absolute N invested in soluble proteins and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis; (15)N flux studies revealed that N for defensive phenolamide synthesis originates from recently assimilated N rather than from RuBisCO turnover. Here we show that a transgenic line of N. attenuata silenced in the expression of RuBisCO (asRUB) invests similar or even larger amounts of N into phenolamide biosynthesis compared with wild type plants, consistent with our previous conclusion that recently assimilated N is channeled into phenolamide synthesis after elicitation. We suggest that the decrease in leaf proteins after simulated herbivory is a tolerance mechanism, rather than a consequence of N-demand for defense biosynthesis.

  10. S-nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata- ribulose-1,5-bisphosphate carboxylase/oxygenase activity targeted for inhibition.

    Science.gov (United States)

    Abat, Jasmeet K; Mattoo, Autar K; Deswal, Renu

    2008-06-01

    Nitric oxide (NO) is a signaling molecule that affects a myriad of processes in plants. However, the mechanistic details are limited. NO post-translationally modifies proteins by S-nitrosylation of cysteines. The soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin switch technique. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, the cytoskeleton, stress and photosynthesis. Some were similar to those previously identified in Arabidopsis thaliana, but kinesin-like protein, glycolate oxidase, putative UDP glucose 4-epimerase and putative DNA topoisomerase II had not been identified as targets previously for any organism. In vitro and in vivo nitrosylation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), one of the targets, was confirmed by immunoblotting. Rubisco plays a central role in photosynthesis, and the effect of S-nitrosylation on its enzymatic activity was determined using NaH14CO3. The NO-releasing compound S-nitrosoglutathione inhibited its activity in a dose-dependent manner suggesting Rubisco inactivation by nitrosylation for the first time.

  11. The metabolism of [3-(13)C]lactate in the rat brain is specific of a pyruvate carboxylase-deprived compartment.

    Science.gov (United States)

    Bouzier, A K; Thiaudiere, E; Biran, M; Rouland, R; Canioni, P; Merle, M

    2000-08-01

    Lactate metabolism in the adult rat brain was investigated in relation with the concept of lactate trafficking between astrocytes and neurons. Wistar rats were infused intravenously with a solution containing either [3-(13)C]lactate (534 mM) or both glucose (750 mM) and [3-(13)C]lactate (534 mM). The time courses of both the concentration and (13)C enrichment of blood glucose and lactate were determined. The data indicated the occurrence of [3-(13)C]lactate recycling through liver gluconeogenesis. The yield of glucose labeling was, however, reduced when using the glucose-containing infusate. After a 20-min or 1-h infusion, perchloric acid extracts of the brain tissue were prepared and subsequently analyzed by (13)C- and (1)H-observed/(13)C-edited NMR spectroscopy. The (13)C labeling of amino acids indicated that [3-(13)C]lactate was metabolized in the brain. Based on the alanine C3 enrichment, lactate contribution to brain metabolism amounted to 35% under the most favorable conditions used. By contrast with what happens with [1-(13)C]glucose metabolism, no difference in glutamine C2 and C3 labeling was evidenced, indicating that lactate was metabolized in a compartment deprived of pyruvate carboxylase activity. This result confirms, for the first time from an in vivo study, that lactate is more specifically a neuronal substrate.

  12. Utilization by the isolated perfused rat liver of N-acetyl-D-(1-/sup 14/C)galactosamine and N-brace/sup 3/H)-acetyl-D-galactosamine for the biosynthesis of glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    MacNicoll, A D; Wusteman, F S; Powell, G M; Curtis, C G [University Coll., Cardiff (UK)

    1978-08-15

    The isolated perfused rat liver system has been used to monitor the utilization of N-(/sup 3/H)acetyl-D-galactosamine and N-acetyl-D-(1-/sup 14/C)galactosamine for the biosynthesis of radiolabeled glycoproteins, which are subsequently secreted into the plasma. Both radiolabels appear in a number of different glycoproteins, predominantly as sialic acid and N-acetylglucosamine. The ratio of labelled sialic acid to labelled N-acetylglucosamine varies for different glycoproteins, but the bulk of N-acetyl-D-galactosamine is incorporated without deacetylation.

  13. Luminal localization of α-tubulin K40 acetylation by cryo-EM analysis of fab-labeled microtubules.

    Directory of Open Access Journals (Sweden)

    Virupakshi Soppina

    Full Text Available The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40 has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

  14. Conversion of 1-alkyl-2-acetyl-sn-glycerols to platelet activating factor and related phospholipids by rabbit platelets

    International Nuclear Information System (INIS)

    Blank, M.L.; Lee, T.; Cress, E.A.; Malone, B.; Fitzgerald, V.; Snyder, F.

    1984-01-01

    The metabolic pathway for 1-alkyl-2-acetyl-sn-glycerols, a recently discovered biologically active neutral lipid class, was elucidated in experiments conducted with rabbit platelets. The total lipid extract obtained from platelets incubated with 1-[1-,2- 3 H]alkyl-2-acetyl-sn-glycerols or 1-alkyl-2-[ 3 H]acetyl-sn-glycerols contained at least six metabolic products. The six metabolites, identified on the basis of chemical and enzymatic reactions combined with thin-layer or high-performance liquid chromatographic analyses, corresponded to 1-alkyl-sn-glycerols, 1-alkyl-2-acetyl-sn-glycero-3-phosphates, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholines, and 1-alkyl-2-actyl-sn-glycero-3-phosphocholines (platelet activating factor). These results indicate that the metabolic pathway for alkylacetylglycerols involves reaction steps catalyzed by the following enzymatic activities: choline- and ethanolamine- phosphotransferases, acetyl-hydrolase, an acyltransferase, and a phosphotransferase. The step responsible for the biosynthesis of platelet activating factor would appear to be the most important reaction in this pathway and this product could explain the hypotensive activities previously described for alkylacetyl-(or propionyl)-glycerols. Of particular interest was the preference exhibited for the utilization of the 1-hexadecyl-2-acetyl-sn-glycerol species in the formation of platelet activating factor

  15. Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells

    International Nuclear Information System (INIS)

    Karaca, Esra; Lewicki, Jakub; Hermanson, Ola

    2015-01-01

    The transcriptional corepressor CtBP2 is essential for proper development of the nervous system. The factor exerts its repression by interacting in complexes with chromatin-modifying factors such as histone deacetylases (HDAC) 1/2 and the histone demethylase LSD1/KDM1. Notably, the histone acetyl transferase p300 acetylates CtBP2 and this is an important regulatory event of the activity and subcellular localization of the protein. We recently demonstrated an essential role for CtBPs as sensors of microenvironmental oxygen levels influencing the differentiation potential of neural stem cells (NSCs), but it is not known whether oxygen levels influence the acetylation levels of CtBP factors. Here we show by using proximity ligation assay (PLA) that CtBP2 acetylation levels increased significantly in undifferentiated, proliferating NSCs under hypoxic conditions. CtBP2 interacted with the class III HDAC Sirt1 but this interaction was unaltered in hypoxic conditions, and treatment with the Sirt1 inhibitor Ex527 did not result in any significant change in total CtBP2 acetylation levels. Instead, we revealed a significant decrease in PLA signal representing CtBP2 dimerization in NSCs under hypoxic conditions, negatively correlating with the acetylation levels. Our results suggest that microenvironmental oxygen levels influence the dimerization and acetylation levels, and thereby the activity, of CtBP2 in proliferating NSCs

  16. Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Karaca, Esra; Lewicki, Jakub; Hermanson, Ola, E-mail: Ola.Hermanson@ki.se

    2015-03-01

    The transcriptional corepressor CtBP2 is essential for proper development of the nervous system. The factor exerts its repression by interacting in complexes with chromatin-modifying factors such as histone deacetylases (HDAC) 1/2 and the histone demethylase LSD1/KDM1. Notably, the histone acetyl transferase p300 acetylates CtBP2 and this is an important regulatory event of the activity and subcellular localization of the protein. We recently demonstrated an essential role for CtBPs as sensors of microenvironmental oxygen levels influencing the differentiation potential of neural stem cells (NSCs), but it is not known whether oxygen levels influence the acetylation levels of CtBP factors. Here we show by using proximity ligation assay (PLA) that CtBP2 acetylation levels increased significantly in undifferentiated, proliferating NSCs under hypoxic conditions. CtBP2 interacted with the class III HDAC Sirt1 but this interaction was unaltered in hypoxic conditions, and treatment with the Sirt1 inhibitor Ex527 did not result in any significant change in total CtBP2 acetylation levels. Instead, we revealed a significant decrease in PLA signal representing CtBP2 dimerization in NSCs under hypoxic conditions, negatively correlating with the acetylation levels. Our results suggest that microenvironmental oxygen levels influence the dimerization and acetylation levels, and thereby the activity, of CtBP2 in proliferating NSCs.

  17. N-acetyl-L-tryptophan, a substance-P receptor antagonist attenuates aluminum-induced spatial memory deficit in rats.

    Science.gov (United States)

    Fernandes, Joylee; Mudgal, Jayesh; Rao, Chamallamudi Mallikarjuna; Arora, Devinder; Basu Mallik, Sanchari; Pai, K S R; Nampoothiri, Madhavan

    2018-06-01

    Neuroinflammation plays an important role in the pathophysiology of Alzheimer's disease. Neurokinin substance P is a key mediator which modulates neuroinflammation through neurokinin receptor. Involvement of substance P in Alzheimer's disease is still plausible and various controversies exist in this hypothesis. Preventing the deleterious effects of substance P using N-acetyl-L-tryptophan, a substance P antagonist could be a promising therapeutic strategy. This study was aimed to evaluate the effect of N-acetyl-L-tryptophan on aluminum induced spatial memory alterations in rats. Memory impairment was induced using aluminum chloride (AlCl 3 ) at a dose of 10 mg/kg for 42 d. After induction of dementia, rats were exposed to 30 and 50 mg/kg of N-acetyl-L-tryptophan for 28 d. Spatial memory alterations were measured using Morris water maze. Acetylcholinesterase activity and antioxidant enzyme glutathione level were assessed in hippocampus, frontal cortex and striatum. The higher dose of N-acetyl-L-tryptophan (50 mg/kg) significantly improved the aluminum induced memory alterations. N-acetyl-L-tryptophan exposure resulted in significant increase in acetylcholinesterase activity and glutathione level in hippocampus. The neuroprotective effect of N-acetyl-L-tryptophan could be due to its ability to block substance P mediated neuroinflammation, reduction in oxidative stress and anti-apoptotic properties. To conclude, N-acetyl-L-tryptophan may be considered as a novel neuroprotective therapy in Alzheimer's disease.

  18. Pathological histone acetylation in Parkinson's disease: Neuroprotection and inhibition of microglial activation through SIRT 2 inhibition.

    Science.gov (United States)

    Harrison, Ian F; Smith, Andrew D; Dexter, David T

    2018-02-14

    Parkinson's disease (PD) is associated with degeneration of nigrostriatal neurons due to intracytoplasmic inclusions composed predominantly of a synaptic protein called α-synuclein. Accumulations of α-synuclein are thought to 'mask' acetylation sites on histone proteins, inhibiting the action of histone acetyltransferase (HAT) enzymes in their equilibrium with histone deacetylases (HDACs), thus deregulating the dynamic control of gene transcription. It is therefore hypothesised that the misbalance in the actions of HATs/HDACs in neurodegeneration can be rectified with the use of HDAC inhibitors, limiting the deregulation of transcription and aiding neuronal homeostasis and neuroprotection in disorders such as PD. Here we quantify histone acetylation in the Substantia Nigra pars compacta (SNpc) in the brains of control, early and late stage PD cases to determine if histone acetylation is a function of disease progression. PD development is associated with Braak-dependent increases in histone acetylation. Concurrently, we show that as expected disease progression is associated with reduced markers of dopaminergic neurons and increased markers of activated microglia. We go on to demonstrate that in vitro, degenerating dopaminergic neurons exhibit histone hypoacetylation whereas activated microglia exhibit histone hyperacetylation. This suggests that the disease-dependent increase in histone acetylation observed in human PD cases is likely a combination of the contributions of both degenerating dopaminergic neurons and infiltrating activated microglia. The HDAC SIRT 2 has become increasingly implicated as a novel target for mediation of neuroprotection in PD: the neuronal and microglial specific effects of its inhibition however remain unclear. We demonstrate that SIRT 2 expression in the SNpc of PD brains remains relatively unchanged from controls and that SIRT 2 inhibition, via AGK2 treatment of neuronal and microglial cultures, results in neuroprotection of

  19. AuBr3-catalyzed azidation of per-O-acetylated and per-O-benzoylated sugars.

    Science.gov (United States)

    Rajput, Jayashree; Hotha, Srinivas; Vangala, Madhuri

    2018-01-01

    Herein we report, for the first time, the successful anomeric azidation of per- O -acetylated and per- O -benzoylated sugars by catalytic amounts of oxophilic AuBr 3 in good to excellent yields. The method is applicable to a wide range of easily accessible per- O -acetylated and per- O -benzoylated sugars. While reaction with per- O -acetylated and per- O -benzoylated monosaccharides was complete within 1-3 h at room temperature, the per- O -benzoylated disaccharides needed 2-3 h of heating at 55 °C.

  20. Acetylation of the pro-apoptotic factor, p53 in the hippocampus following cerebral ischemia and modulation by estrogen.

    Directory of Open Access Journals (Sweden)

    Limor Raz

    Full Text Available Recent studies demonstrate that acetylation of the transcription factor, p53 on lysine(373 leads to its enhanced stabilization/activity and increased susceptibility of cells to stress. However, it is not known whether acetylation of p53 is altered in the hippocampus following global cerebral ischemia (GCI or is regulated by the hormone, 17β-estradiol (17β-E(2, and thus, this study examined these issues.The study revealed that Acetyl p53-Lysine(373 levels were markedly increased in the hippocampal CA1 region after GCI at 3 h, 6 h and 24 h after reperfusion, an effect strongly attenuated by 17β-E(2. 17β-E(2 also enhanced interaction of p53 with the ubiquitin ligase, Mdm2, increased ubiquitination of p53, and induced its down-regulation, as well as attenuated elevation of the p53 transcriptional target, Puma. We also observed enhanced acetylation of p53 at a different lysine (Lys(382 at 3 h after reperfusion, and 17β-E(2 also markedly attenuated this effect. Furthermore, administration of an inhibitor of CBP/p300 acetyltransferase, which acetylates p53, was strongly neuroprotective of the CA1 region following GCI. In long-term estrogen deprived (LTED animals, the ability of 17β-E(2 to attenuate p53 acetylation was lost, and intriguingly, Acetyl p53-Lysine(373 levels were markedly elevated in sham (non-ischemic LTED animals. Finally, intracerebroventricular injections of Gp91ds-Tat, a specific NADPH oxidase (NOX2 inhibitor, but not the scrambled tat peptide control (Sc-Tat, attenuated acetylation of p53 and reduced levels of Puma following GCI.The studies demonstrate that p53 undergoes enhanced acetylation in the hippocampal CA1 region following global cerebral ischemia, and that the neuroprotective agent, 17β-E(2, markedly attenuates the ischemia-induced p53 acetylation. Furthermore, following LTED, the suppressive effect of 17β-E(2 on p53 acetylation is lost, and p53 acetylation increases in the hippocampus, which may explain previous

  1. E2F family members are differentially regulated by reversible acetylation

    DEFF Research Database (Denmark)

    Marzio, G; Wagener, C; Gutierrez, M I

    2000-01-01

    of the other E2F family members. Here we report that E2F-1, -2, and -3, but not E2F-4, -5, and -6, associate with and are acetylated by p300 and cAMP-response element-binding protein acetyltransferases. Acetylation occurs at three conserved lysine residues located at the N-terminal boundary of their DNA......The six members of the E2F family of transcription factors play a key role in the control of cell cycle progression by regulating the expression of genes involved in DNA replication and cell proliferation. E2F-1, -2, and -3 belong to a structural and functional subfamily distinct from those...

  2. Cigarette Smoking, N-Acetyltransferase 2 Acetylation Status, and Bladder Cancer Risk

    DEFF Research Database (Denmark)

    Marcus, P.M.; Hayes, R.B.; Vineis, P.

    2000-01-01

    Tobacco use is an established cause of bladder cancer. The ability to detoxify aromatic amines, which are present in tobacco and are potent bladder carcinogens, is compromised in persons with the N-acetyltransferase 2 slow acetylation polymorphism. The relationship of cigarette smoking with bladder...... cancer risk therefore has been hypothesized to be stronger among slow acetylators. The few studies to formally explore such a possibility have produced inconsistent results, however. To assess this potential gene-environment interaction in as many bladder cancer studies as possible and to summarize...... results, we conducted a meta-analysis using data from 16 bladder cancer studies conducted in the general population (n = 1999 cases), Most had been conducted in European countries. Because control subjects were unavailable for a number of these studies, we used a case-series design, which can be used...

  3. Hypochlorite-mediated fragmentation of hyaluronan, chondroitin sulfates, and related N-acetyl glycosamines

    DEFF Research Database (Denmark)

    Rees, Martin D; Hawkins, Clare Louise; Davies, Michael Jonathan

    2003-01-01

    Myeloperoxidase released from activated phagocytes reacts with H(2)O(2) in the presence of chloride ions to give hypochlorous acid. This oxidant has been implicated in the fragmentation of glycosaminoglycans, such as hyaluronan and chondroitin sulfates. In this study it is shown that reaction...... processes. In the case of glycosaminoglycan-derived amidyl radicals, evidence has been obtained in studies with model glycosides that these radicals undergo rapid intramolecular abstraction reactions to give carbon-centered radicals at C-2 on the N-acetyl glycosamine rings (via a 1,2-hydrogen atom shift......) and at C-4 on the neighboring uronic acid residues (via 1,5-hydrogen atom shifts). The C-4 carbon-centered radicals, and analogous species derived from model glycosides, undergo pH-independent beta-scission reactions that result in glycosidic bond cleavage. With N-acetyl glucosamine C-1 alkyl glycosides...

  4. Hybrid Organometallic-Inorganic Nanomaterial: Acetyl Ferrocene Schiff base Immobilized on Silica Coated Magnetite Nanoparticles

    Directory of Open Access Journals (Sweden)

    M. Masteri-Farahani

    2015-10-01

    Full Text Available In  this  work,  a  new  hybrid  organometallic-inorganic  hybrid nanomaterial was prepared by immobilization of acetyl ferrocene on the  surface  of magnetite  nanoparticles. Covalent  grafting of silica coated magnetite nanoparticles (SCMNPs with 3-aminopropyl triethoxysilane gave aminopropyl-modified magnetite nanoparticles (AmpSCMNPs. Then, Schiff base condensation  of AmpSCMNPs with acetyl  ferrocene resulted in the preparation of acferro-SCMNPs hybrid nanomaterial. Characterization of the prepared nanomaterial was performed with different physicochemical methods such as Fourier transform infrared spectroscopy (FT-IR, X-ray diffraction (XRD, vibrating sample magnetometry (VSM, thermogravimetric analysis (TGA, scanning electron microscopy (SEM, and transmission electron microscopy (TEM. VSM analysis showed superparamagnetic properties of the prepared nanomaterial and TEM and SEM analyses indicated the relatively spherical nanoparticles with 15 nm average size.

  5. The Role of Pectin Acetylation in the Organization of Plant Cell Walls

    DEFF Research Database (Denmark)

    Fimognari, Lorenzo

    adopt defined 3D organization to allow their composition/interactions to be tweaked upon developmental need. Failure to build functional cell wall architecture will affect plant growth and resistance to stresses. In this PhD dissertation I explored the role of pectin acetylation in controlling...... wall organization, namely polysaccharides-to-polysaccharides interactions. These results suggest that cell wall acetylation is a mechanism that plants evolved to control cell wall organization. In Manuscript III, we report the characterization of Arabidopsis mutants trichome birefringence like (tbl) 10......All plant cells are surrounded by one or more cell wall layers. The cell wall serves as a stiff mechanical support while it allows cells to expand and provide a protective barrier to invading pathogens. Cell walls are dynamic structures composed of entangled cell wall polysaccharides that must...

  6. Synthesis, crystal and supramolecular structure of rac-N-acetyl-2- thiohydantoin-asparagine

    Directory of Open Access Journals (Sweden)

    Gerzon E. Delgado

    2014-05-01

    Full Text Available The title compound, C7H9N3O3S, also known as rac-N-acetyl-5-propionamide-2-thioxo-imidazolidin-4-one, crystallize in the monoclinic system with space group P21/n (Nº14, Z=4, and unit cell parameters a= 9.338 (7 Å, b= 7.545 (5 Å, c= 13.212 (10 Å, E= 97.10 (2°, V= 932.8 (12 Å3. The acetyl group and the mean plane of the ureido group form an angle of 81.0 (2°. In the supramolecular structure, the molecules are joined by N--H···O hydrogen bonds into cyclic structures with graph-set R2 2(14 and R2 2(16, forming a three-dimensional network.

  7. Isolation and pharmacological screening of 8-O-acetyl harpagide from Ajuga bracteosa wall

    International Nuclear Information System (INIS)

    Shafi, N.; Khan, G.A.; Ahmad, K.D.; Gilani, N.D.; Arfan, M.

    2004-01-01

    8-O-Acetyl harpagide was isolated and characterized from Jaguar bracteosa Wall, a species indigenous to Pakistan. Pharmacological screening of the compound for antibacterial, antifungal, antispasmodic, cardiotonic and antipyretic activities was carried out. The compound was found effective against a number of human pathogenic bacteria and fungi. Antispasmodic and cardiotonic effects elicited by the compound were also found. The compound also exhibited antipyretic activity when administered in the higher doses. (author)

  8. Characterization of FIBCD1 as an acetyl group-binding receptor that binds chitin

    DEFF Research Database (Denmark)

    Schlosser, Anders; Thomsen, Theresa; Moeller, Jesper B

    2009-01-01

    Chitin is a highly acetylated compound and the second most abundant biopolymer in the world next to cellulose. Vertebrates are exposed to chitin both through food ingestion and when infected with parasites, and fungi and chitin modulate the immune response in different directions. We have...... fragments. FIBCD1 may play an important role in controlling the exposure of intestine to chitin and chitin fragments, which is of great relevance for the immune defense against parasites and fungi and for immune response modulation....

  9. N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor1[S

    OpenAIRE

    Zhang, Hao; Jing, Xigang; Shi, Yang; Xu, Hao; Du, Jianhai; Guan, Tongju; Weihrauch, Dorothee; Jones, Deron W.; Wang, Weiling; Gourlay, David; Oldham, Keith T.; Hillery, Cheryl A.; Pritchard, Kirkwood A.

    2013-01-01

    Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (⩽4,000 μM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HO...

  10. PROCESS OPTIMIZATION OF TETRA ACETYL ETHYLENE DIAMINE ACTIVATED HYDROGEN PEROXIDE BLEACHING OF POPULUS NIGRA CTMP

    OpenAIRE

    Qiang Zhao; Junwen Pu; Shulei Mao; Guibo Qi

    2010-01-01

    To enhance the bleaching efficiency, the activator of tetra acetyl ethylene diamine (TAED) was used in conventional H2O2 bleaching. The H2O2/TAED bleaching system can accelerate the reaction rate and shorten bleaching time at relative low temperature, which can reduce the production cost. In this research, the process with hydrogen peroxide activated by TAED bleaching of Populus nigra chemi-thermo mechanical pulp was optimized. Suitable bleaching conditions were confirmed as follows: pulp con...

  11. Efficient and reproducible synthesis of [1-{sup 11}C]acetyl chloride using the loop method

    Energy Technology Data Exchange (ETDEWEB)

    Arai, Takuya [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Zhang, Ming-Rong [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)], E-mail: zhang@nirs.go.jp; Ogawa, Masanao [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); SHI Accelerator Service Co. Ltd., 1-17-6 Osaki, Shinagawa-ku, Tokyo 141-8686 (Japan); Fukumura, Toshimitsu; Kato, Koichi; Suzuki, Kazutoshi [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2009-02-15

    [1-{sup 11}C]Acetyl chloride ([{sup 11}C]AcCl), an important [{sup 11}C]acylating agent, was synthesized by reacting [{sup 11}C]CO{sub 2} with methylmagnesium bromide coated on the inner surface of a polyethylene loop (loop method). By optimizing the reaction conditions and synthesis parameters, [1-{sup 11}C]phenylacetate and [1-{sup 11}C]benzylacetate were produced from [{sup 11}C]AcCl in high radiochemical yield and specific activity.

  12. Distinct patterns of histone methylation and acetylation in human interphase nuclei

    Czech Academy of Sciences Publication Activity Database

    Skalníková, M.; Bártová, Eva; Ulman, V.; Matula, P.; Svoboda, D.; Harničarová, Andrea; Kozubek, Michal; Kozubek, Stanislav

    2007-01-01

    Roč. 56, č. 6 (2007), s. 797-806 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA204/06/0978; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : histone methylation * acetylation * X chromosome Subject RIV: BO - Biophysics Impact factor: 1.505, year: 2007

  13. Synthesis and antiproliferative activity of diethyl 5- acetyl-4-methyl- 6 ...

    African Journals Online (AJOL)

    Diethyl 5-acetyl-4-methyl-6-(2-fluorophenylimino)-6H-thiopyran-2,3-dicarboxylate (3TM) was synthesized and the antiproliferative activity of 3TM is reported here. Compound 3TM inhibits the growth of human colon cancer HCT-15 with an IC50 value of 4.5 μM and breast cancer MCF-7 with an IC50 value of 7 μM in a ...

  14. Antiproliferative effects of TSA, PXD‑101 and MS‑275 in A2780 and MCF7 cells: Acetylated histone H4 and acetylated tubulin as markers for HDACi potency and selectivity.

    Science.gov (United States)

    Androutsopoulos, Vasilis P; Spandidos, Demetrios A

    2017-12-01

    Inhibition of histone deacetylase enzymes (HDACs) has been well documented as an attractive target for the development of chemotherapeutic drugs. The present study investigated the effects of two prototype hydroxamic acid HDAC inhibitors, namely Trichostatin A (TSA) and Belinostat (PXD‑101) and the benzamide Entinostat (MS‑275) in A2780 ovarian carcinoma and MCF7 breast adenocarcinoma cells. The three HDACi inhibited the proliferation of A2780 and MCF7 cells at comparable levels, below the µM range. Enzyme inhibition assays in a cell‑free system showed that TSA was the most potent inhibitor of total HDAC enzyme activity followed by PXD‑101 and MS‑275. Incubation of A2780 and MCF7 cells with the hydroxamates TSA and PXD‑101 for 24 h resulted in a dramatic increase of acetylated tubulin induction (up to 30‑fold for TSA). In contrast to acetylated tubulin, western blot analysis and flow cytometry indicated that the induction of acetylated histone H4 was considerably smaller. The benzamide MS‑275 exhibited nearly a 2‑fold induction of acetylated histone H4 and an even smaller induction of acetylated tubulin in A2780 and MCF7 cells. Taken together, these data suggest that although the three HDACi were equipotent in inhibiting proliferation of MCF7 and A2780 cells, only the benzamide MS‑275 did not induce acetylated tubulin expression, a marker of class IIb HDACs.

  15. Structural and functional features of lysine acetylation of plant and animal tubulins.

    Science.gov (United States)

    Rayevsky, Alexey V; Sharifi, Mohsen; Samofalova, Dariya A; Karpov, Pavel A; Blume, Yaroslav B

    2017-10-10

    The study of the genome and the proteome of different species and representatives of distinct kingdoms, especially detection of proteome via wide-scaled analyses has various challenges and pitfalls. Attempts to combine all available information together and isolate some common features for determination of the pathway and their mechanism of action generally have a highly complicated nature. However, microtubule (MT) monomers are highly conserved protein structures, and microtubules are structurally conserved from Homo sapiens to Arabidopsis thaliana. The interaction of MT elements with microtubule-associated proteins and post-translational modifiers is fully dependent on protein interfaces, and almost all MT modifications are well described except acetylation. Crystallography and interactome data using different approaches were combined to identify conserved proteins important in acetylation of microtubules. Application of computational methods and comparative analysis of binding modes generated a robust predictive model of acetylation of the ϵ-amino group of Lys40 in α-tubulins. In turn, the model discarded some probable mechanisms of interaction between elements of interest. Reconstruction of unresolved protein structures was carried out with modeling by homology to the existing crystal structure (PDBID: 1Z2B) from B. taurus using Swiss-model server, followed by a molecular dynamics simulation. Docking of the human tubulin fragment with Lys40 into the active site of α-tubulin acetyltransferase, reproduces the binding mode of peptidomimetic from X-ray structure (PDBID: 4PK3). © 2017 International Federation for Cell Biology.

  16. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    Science.gov (United States)

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L.; Huber, Steven C.; Zhao, Youfu

    2015-01-01

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

  17. Acetylation of oil palm empty fruit bunch fiber as an adsorbent for removal of crude oil.

    Science.gov (United States)

    Asadpour, Robabeh; Sapari, Nasiman B; Isa, Mohamed Hasnain; Kakooei, Saeid

    2016-06-01

    Removal of oil spillage from the environment is a global concern. Various methods, including the use of fibers as sorbents, have been developed for oil spill control. Oil palm empty fruit bunch (OPEFB) fiber is a plant biomass that may be acetylated by acetic anhydride using N-bromosuccinimide (NBS) as a catalyst; here, the extent of acetylation may be calculated in terms of weight percent gain (WPG). The modified fiber was used to remove Tapis and Arabian crude oils. The optimum time, temperature, and catalyst concentration were 4 h, 120 °C, and 3 %, respectively, and these parameters could achieve an 11.49 % increase in WPG. The optimized parameters improved the adsorption capacity of OPEFB fibers for crude oil removal. The acetylated OPEFB fibers were characterized by using Fourier transform infrared spectroscopy and field emission scanning electron microscopy to observe the functional groups available and morphology. Kinetic and isotherm studies were conducted using different contact times and oil/water ratios. The rate of oil sorption onto the OPEFB fibers can be adequately described by the pseudo-second-order equation. Adsorption studies revealed that adsorption of crude oil on treated OPEFB fiber could be best described by the Langmuir isotherm model.

  18. Copper(II) Binding Sites in N-Terminally Acetylated α-Synuclein: A Theoretical Rationalization.

    Science.gov (United States)

    Ramis, Rafael; Ortega-Castro, Joaquín; Vilanova, Bartolomé; Adrover, Miquel; Frau, Juan

    2017-08-03

    The interactions between N-terminally acetylated α-synuclein and Cu(II) at several binding sites have been studied with DFT calculations, specifically with the M06 hybrid functional and the ωB97X-D DFT-D functional. In previous experimental studies, Cu(II) was shown to bind several α-synuclein residues, including Met1-Asp2 and His50, forming square planar coordination complexes. Also, it was determined that a low-affinity binding site exists in the C-terminal domain, centered on Asp121. However, in the N-terminally acetylated protein, present in vivo, the Met1 site is blocked. In this work, we simplify the representation of the protein by modeling each experimentally found binding site as a complex between an N-terminally acetylated α-synuclein dipeptide (or several independent residues) and a Cu(II) cation, and compare the results with a number of additional, structurally analogous sites not experimentally found. This way of representing the binding sites, although extremely simple, allows us to reproduce experimental results and to provide a theoretical rationale to explain the preference of Cu(II) for certain sites, as well as explicit geometrical structures for the complexes formed. These results are important to understand the interactions between α-synuclein and Cu(II), one of the factors inducing structural changes in the protein and leading to aggregated forms of it which may play a role in neurodegeneration.

  19. Acetylation of the Cd8 Locus by KAT6A Determines Memory T Cell Diversity

    Directory of Open Access Journals (Sweden)

    Dane M. Newman

    2016-09-01

    Full Text Available How functionally diverse populations of pathogen-specific killer T cells are generated during an immune response remains unclear. Here, we propose that fine-tuning of CD8αβ co-receptor levels via histone acetylation plays a role in lineage fate. We show that lysine acetyltransferase 6A (KAT6A is responsible for maintaining permissive Cd8 gene transcription and enabling robust effector responses during infection. KAT6A-deficient CD8+ T cells downregulated surface CD8 co-receptor expression during clonal expansion, a finding linked to reduced Cd8α transcripts and histone-H3 lysine 9 acetylation of the Cd8 locus. Loss of CD8 expression in KAT6A-deficient T cells correlated with reduced TCR signaling intensity and accelerated contraction of the effector-like memory compartment, whereas the long-lived memory compartment appeared unaffected, a result phenocopied by the removal of the Cd8 E8I enhancer element. These findings suggest a direct role of CD8αβ co-receptor expression and histone acetylation in shaping functional diversity within the cytotoxic T cell pool.

  20. VUV photoionization and dissociative photoionization of the prebiotic molecule acetyl cyanide: Theory and experiment

    International Nuclear Information System (INIS)

    Bellili, A.; Hochlaf, M.; Schwell, M.; Bénilan, Y.; Fray, N.; Gazeau, M.-C.; Mogren Al-Mogren, M.; Guillemin, J.-C.; Poisson, L.

    2014-01-01

    The present combined theoretical and experimental investigation concerns the single photoionization of gas-phase acetyl cyanide and the fragmentation pathways of the resulting cation. Acetyl cyanide (AC) is inspired from both the chemistry of cyanoacetylene and the Strecker reaction which are thought to be at the origin of medium sized prebiotic molecules in the interstellar medium. AC can be formed by reaction from cyanoacetylene and water but also from acetaldehyde and HCN or the corresponding radicals. In view of the interpretation of vacuum ultraviolet (VUV) experimental data obtained using synchrotron radiation, we explored the ground potential energy surface (PES) of acetyl cyanide and of its cation using standard and recently implemented explicitly correlated methodologies. Our PES covers the regions of tautomerism (between keto and enol forms) and of the lowest fragmentation channels. This allowed us to deduce accurate thermochemical data for this astrobiologically relevant molecule. Unimolecular decomposition of the AC cation turns out to be very complex. The implications for the evolution of prebiotic molecules under VUV irradiation are discussed

  1. The heat of formation of the acetyl cation: a theoretical evaluation

    Science.gov (United States)

    Smith, Brian J.; Radom, Leo

    1990-12-01

    Ab initio molecular orbital calculations have been used to obtain the heat of formation of the acetyl cation. In one set of calculations, the reverse activation barrier for the production of acetyl cation from acetaldehyde has been shown to be significantly different zero and the value obtained (9.8 kJ mol-1 at 298 K) has been used to correct the [Delta]Hof298 (CH3CO+) value derived from appearance energy measurements. In a second set of calculations, [Delta]H°f298 (CH3CO+) has been obtained from the calculated heats of a number of reactions involving the acetyl cation together with experimental heats of formation for the species involved. The best theoretical estimate for [Delta]H°f298 (CH3CO+), obtained as a mean of results from the two approaches, is 658 kJ mol-1. The best theoretical estimate for [Delta]H°f0(CH3CO+), obtained in a similar manner, is 665 kJ mol-1.

  2. Infrared spectroscopy of the acetyl cation and its protonated ketene isomer

    Science.gov (United States)

    Mosley, J. D.; Young, J. W.; Duncan, M. A.

    2014-07-01

    [C2,H3,O]+ ions are generated with a pulsed discharge in a supersonic expansion containing methyl acetate or acetone. These ions are mass selected and their infrared spectra are recorded via laser photodissociation and the method of argon tagging. Computational chemistry is employed to investigate structural isomers and their spectra. The acetyl cation (CH3CO+) is the global minimum and protonated ketene (CH2COH+) is the next lowest energy isomer (+176.2 kJ/mol). When methyl acetate is employed as the precursor, the infrared spectrum reveals that only the acetyl cation is formed. Partially resolved rotational structure reveals rotation about the C3 axis. When acetone is used as the precursor, acetyl is still the most abundant cation, but there is also a minor component of protonated ketene. Computations reveal a significant barrier to interconversion between the two isomers (+221 kJ/mol), indicating that protonated ketene must be obtained via kinetic trapping. Both isomers may be present in interstellar environments, and their implications for astrochemistry are discussed.

  3. Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

    Science.gov (United States)

    Bergmann, Jan H; Jakubsche, Julia N; Martins, Nuno M; Kagansky, Alexander; Nakano, Megumi; Kimura, Hiroshi; Kelly, David A; Turner, Bryan M; Masumoto, Hiroshi; Larionov, Vladimir; Earnshaw, William C

    2012-01-15

    Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity--kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

  4. Protective Roles of N-acetyl Cysteine and/or Taurine against Sumatriptan-Induced Hepatotoxicity

    Directory of Open Access Journals (Sweden)

    Javad Khalili Fard

    2016-12-01

    Full Text Available Purpose: Triptans are the drug category mostly prescribed for abortive treatment of migraine. Most recent cases of liver toxicity induced by triptans have been described, but the mechanisms of liver toxicity of these medications have not been clear. Methods: In the present study, we obtained LC50 using dose-response curve and investigated cell viability, free radical generation, lipid peroxide production, mitochondrial injury, lysosomal membrane damage and the cellular glutathione level as toxicity markers as well as the beneficial effects of taurine and/or N-acetyl cysteine in the sumatriptan-treated rat parenchymal hepatocytes using accelerated method of cytotoxicity mechanism screening. Results: It was revealed that liver toxicity induced by sumatriptan in in freshly isolated parenchymal hepatocytes is dose-dependent. Sumatriptan caused significant free radical generation followed by lipid peroxide formation, mitochondrial injury as well as lysosomal damage. Moreover, sumatriptan reduced cellular glutathione content. Taurine and N-acetyl cysteine were able to protect hepatocytes against sumatriptan-induced harmful effects. Conclusion: It is concluded that sumatriptan causes oxidative stress in hepatocytes and the decreased hepatocytes glutathione has a key role in the sumatriptan-induced harmful effects. Also, N-acetyl cysteine and/or taurine could be used as treatments in sumatriptan-induced side effects.

  5. RNA content in the nucleolus alters p53 acetylation via MYBBP1A

    Science.gov (United States)

    Kuroda, Takao; Murayama, Akiko; Katagiri, Naohiro; Ohta, Yu-mi; Fujita, Etsuko; Masumoto, Hiroshi; Ema, Masatsugu; Takahashi, Satoru; Kimura, Keiji; Yanagisawa, Junn

    2011-01-01

    A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53–p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity. PMID:21297583

  6. VUV photoionization and dissociative photoionization of the prebiotic molecule acetyl cyanide: Theory and experiment

    Energy Technology Data Exchange (ETDEWEB)

    Bellili, A.; Hochlaf, M., E-mail: hochlaf@univ-mlv.fr, E-mail: martin.schwell@lisa.u-pec.fr [Laboratoire Modélisation et Simulation Multi Echelle, MSME UMR 8208 CNRS, Université Paris-Est, 5 bd Descartes, 77454 Marne-la-Vallée (France); Schwell, M., E-mail: hochlaf@univ-mlv.fr, E-mail: martin.schwell@lisa.u-pec.fr; Bénilan, Y.; Fray, N.; Gazeau, M.-C. [Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), UMR 7583 CNRS, Institut Pierre et Simon Laplace, Universités Paris-Est Créteil et Paris Diderot, 61 Avenue du Général de Gaulle, 94010 Créteil (France); Mogren Al-Mogren, M. [Chemistry Department, Faculty of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Guillemin, J.-C. [Institut des Sciences Chimiques de Rennes, Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, Allée de Beaulieu, CS 50837, 35708 Rennes Cedex 7 (France); Poisson, L. [Laboratoire Francis Perrin, CNRS URA 2453, CEA, IRAMIS, Laboratoire Interactions Dynamique et Lasers, Bât 522, F-91191 Gif/Yvette (France)

    2014-10-07

    The present combined theoretical and experimental investigation concerns the single photoionization of gas-phase acetyl cyanide and the fragmentation pathways of the resulting cation. Acetyl cyanide (AC) is inspired from both the chemistry of cyanoacetylene and the Strecker reaction which are thought to be at the origin of medium sized prebiotic molecules in the interstellar medium. AC can be formed by reaction from cyanoacetylene and water but also from acetaldehyde and HCN or the corresponding radicals. In view of the interpretation of vacuum ultraviolet (VUV) experimental data obtained using synchrotron radiation, we explored the ground potential energy surface (PES) of acetyl cyanide and of its cation using standard and recently implemented explicitly correlated methodologies. Our PES covers the regions of tautomerism (between keto and enol forms) and of the lowest fragmentation channels. This allowed us to deduce accurate thermochemical data for this astrobiologically relevant molecule. Unimolecular decomposition of the AC cation turns out to be very complex. The implications for the evolution of prebiotic molecules under VUV irradiation are discussed.

  7. Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots

    Science.gov (United States)

    Wu, Zhen; Fallahi, Mohammad; Ouizem, Souad; Liu, Qin; Li, Weimin; Costi, Roberta; Roush, William R.; Bois, Philippe R. J.

    2016-01-01

    ABSTRACT Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined “hot spots.” In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots. PMID:27821479

  8. Carboxamide SIRT1 inhibitors block DBC1 binding via an acetylation-independent mechanism

    Science.gov (United States)

    Hubbard, Basil P; Loh, Christine; Gomes, Ana P; Li, Jun; Lu, Quinn; Doyle, Taylor LG; Disch, Jeremy S; Armour, Sean M; Ellis, James L; Vlasuk, George P; Sinclair, David A

    2013-01-01

    SIRT1 is an NAD+-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1. PMID:23892437

  9. Curcumin-induced histone acetylation inhibition improves stress-induced gastric ulcer disease in rats.

    Science.gov (United States)

    He, Ping; Zhou, Renmin; Hu, Guorui; Liu, Zhifeng; Jin, Yu; Yang, Guang; Li, Mei; Lin, Qian

    2015-03-01

    Curcumin is known to possess anti‑inflammatory properties. Despite the fact that curcumin is known to be a strong inhibitor of H+, K+‑ATPase activity, the mechanism underlying the curcumin‑induced inhibition of the transcription of the H+, K+‑ATPase α subunit in gastric mucosal parietal cells remains unclear. The present study investigated the possible mechanism by which curcumin inhibits stomach H+, K+‑ATPase activity during the acute phase of gastric ulcer disease. A rat model of stress‑induced gastric ulcers was produced, in which the anti‑ulcer effects of curcumin were examined. Curcumin‑induced inhibition of the H+, K+‑ATPase promoter via histone acetylation, was verified using a chromatin immunoprecipitation assay. The results showed that curcumin improved stress‑induced gastric ulcer disease in rats, as demonstrated by increased pH values and reduced gastric mucosal hemorrhage and ulcer index. These effects were accompanied by a significant reduction in the level of histone H3 acetylation at the site of the H+, K+‑ATPase promoter and in the expression of the gastric H+,K+‑ATPase α subunit gene and protein. In conclusion, curcumin downregulated the acetylation of histone H3 at the site of the H+, K+‑ATPase promoter gene, thereby inhibiting the transcription and expression of the H+, K+‑ATPase gene. Curcumin was shown to have a preventive and therapeutic effect in gastric ulcer disease.

  10. Inhibition of FoxO1 acetylation by INHAT subunit SET/TAF-Iβ induces p21 transcription.

    Science.gov (United States)

    Chae, Yun-Cheol; Kim, Kee-Beom; Kang, Joo-Young; Kim, Se-Ryeon; Jung, Hyeon-Soo; Seo, Sang-Beom

    2014-08-25

    Post-translational modification of forkhead family transcription factor, FoxO1, is an important regulatory mode for its diverse activities. FoxO1 is acetylated by HAT coactivators and its transcriptional activity is decreased via reduced DNA binding affinity. Here, we report that SET/TAF-Iβ inhibited p300-mediated FoxO1 acetylation in an INHAT domain-dependent manner. SET/TAF-Iβ interacted with FoxO1 and activated transcription of FoxO1 target gene, p21. Moreover, SET/TAF-Iβ inhibited acetylation of FoxO1 and increased p21 transcription induced by oxidative stress. Our results suggest that SET/TAF-Iβ inhibits FoxO1 acetylation and activates its transcriptional activity toward p21. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Processing of mutant N-acetyl-alpha-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature

    NARCIS (Netherlands)

    Meijer, O. L. M.; te Brinke, H.; Ofman, R.; IJlst, L.; Wijburg, F. A.; van Vlies, N.

    2017-01-01

    Background: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU), resulting in accumulation of heparan sulfate. The disease spectrum comprises a severe, rapidly

  12. Impact of high glucose concentration on aspirin-induced acetylation of human serum albumin: An in vitro study

    Directory of Open Access Journals (Sweden)

    Francesco Finamore

    2014-06-01

    Full Text Available Aspirin (ASA plays a key role in protecting high risk cardiovascular patients from ischaemic events. The modifications underlying its effects are the results of the trans-acetylation that occurs between ASA and the amino groups made up of lysine and N-terminal residues. ASA's effects have also been demonstrated on several plasma proteins, including human serum albumin (HSA. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair ASA's acetylation process. Using immunoblotting and mass spectrometry, this study characterized the degree of HSA acetylation mediated by ASA in vitro, as well as the impact of high glucose concentrations. Glycation's influence on HSA acetylation might impair the latter's biological functions, leading to a potential failure of ASA to prevent cardiovascular complications in diabetes.

  13. Structure of 1,5-Anhydro-D-Fructose: X-ray Analysis of Crystalline Acetylated Dimeric Forms

    DEFF Research Database (Denmark)

    Lundt, Inge; Andersen, Søren Møller; Marcussen, Jan

    1998-01-01

    Acetylation of 1,5-anhydro-D-fructose under acidic conditions gave two crystalline acetylated dimeric forms, which by X-ray analysis were shown to be diastereomeric spiroketals formed between C-2 and C-2´/C-3´. The structures of the compounds differed only at the configuration at C-2. Acetylation...... or benzoylation of 1,5-anhydro-D-fructose in pyridine yielded 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-enos-2-ulopyra -nose or crystalline 1,5-anhydro-3,6-di-O-benzoyl-4-deoxy-D-glycero-hex-3-enos-2-ulo-py ranose....

  14. Characterization of moisture in acetylated and propionylated radiata pine using low-field nuclear magnetic resonance (LFNMR) relaxometry

    DEFF Research Database (Denmark)

    Beck, Greeley; Thybring, Emil Engelund; Thygesen, Lisbeth Garbrecht

    2018-01-01

    . A possible explanation is the counteracting effects of decreased hydrophilicity and reduced moisture content (MC) of these water populations at higher levels of acetylation. The evaluation of propionylation on WCW T2 data was complicated by peak splitting in the relaxation spectrum. Constant T2 values......Moisture in radiata pine (Pinus radiata D. Don) earlywood (EW), which was acetylated or propionylated to various degrees, was measured by low-field nuclear magnetic resonance (LFNMR) relaxometry. Spin-spin relaxation times (T2) were determined for fully saturated samples at 22 and -18°C. T2 values...... for EW lumen water increased with increasing acetylation weight percentage gain (WPG), perhaps caused by the less hydrophilic acetylated wood (AcW) surface. Cell wall water (WCW) and the water in pits and small voids also showed increasing T2 values as a function of WPG but with a weaker tendency...

  15. Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate. [Spinacia oleracea

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Genhai; Jensen, R.G. (Univ. of Arizona, Tucson (USA))

    1990-05-01

    The properties of the tight and specific binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO{sub 2} and Mg{sup 2+}, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of ({sup 14}C)CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported. The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg{sup 2+}. Addition of ({sup 14}C)CABP and EDTA stopped binding of Mg{sup 2+} and allowed tight binding of the radiolabel only to sites which were CO{sub 2}/Mg{sup 2+}-activated at that moment. The rate of CO{sub 2} fixation was proportional to the CO{sub 2}/Mg{sup 2+}-activated sites. During light-dependent CO{sub 2} fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg{sup 2+} and CO{sub 2}. Lysis of chloroplasts in media with ({sup 14}C)CABP plus EDTA estimated those carbamylated sites having Mg{sup 2+}. The loss of Rubisco activation during illumination was partially due to the lack of Mg{sup 2+} to stabilize the carbamylated sites.

  16. Cinnamic acid-inhibited ribulose-1,5-bisphosphate carboxylase activity is mediated through decreased spermine and changes in the ratio of polyamines in cowpea.

    Science.gov (United States)

    Huang, Xingxue; Bie, Zhilong

    2010-01-01

    This study investigated the effects of cinnamic acid (CA) on ribulose-1,5-bisphosphate carboxylase (RuBPC) activity and the endogenous polyamine levels of cowpea leaves. The results show that 0.1 mM CA treatment decreased photosynthetic rate (P(n)) and RuBPC activity, but it did not affect the maximal photochemical efficiency of PSII (F(v)/F(m)), the actual photochemical efficiency of PSII (PhiPSII), intercellular CO(2) concentration (C(i)), and relative chlorophyll content. These suggest that the decrease in P(n) is at least partially attributed to a lowered RuBPC activity. In addition, 0.1 mM CA treatment increased the putrescine (Put) level, but decreased spermidine (Spd) and spermine (Spm) levels, thereby reducing the (Spd+Spm)/Put (PAs) ratio in the leaves. The exogenous application of 1 mM Spd markedly reversed these CA-induced effects for polyamine and partially restored the PAs ratio and RuBPC activity in leaves. Methylglyoxal-bis (guanylhydrazone) (MGBG), which is an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), results in the inability of activated cells to synthesize Spd and exacerbates the negative effects induced by CA. The exogenous application of 1 mM D-arginine (D-Arg), which is an inhibitor of Put biosynthesis, decreased the levels of Put, but increased the PAs ratio and RuBPC activity in leaves. These results suggest that 0.1 mM CA inhibits RuBPC activity by decreasing the levels of endogenous free and perchloric acid soluble (PS) conjugated Spm, as well as the PAs ratio.

  17. Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies.

    Science.gov (United States)

    Prins, Anneke; van Heerden, Philippus D R; Olmos, Enrique; Kunert, Karl J; Foyer, Christine H

    2008-01-01

    The roles of cysteine proteinases (CP) in leaf protein accumulation and composition were investigated in transgenic tobacco (Nicotiana tabacum L.) plants expressing the rice cystatin, OC-1. The OC-1 protein was present in the cytosol, chloroplasts, and vacuole of the leaves of OC-1 expressing (OCE) plants. Changes in leaf protein composition and turnover caused by OC-1-dependent inhibition of CP activity were assessed in 8-week-old plants using proteomic analysis. Seven hundred and sixty-five soluble proteins were detected in the controls compared to 860 proteins in the OCE leaves. A cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase, and two ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase isoforms were markedly altered in abundance in the OCE leaves. The senescence-related decline in photosynthesis and Rubisco activity was delayed in the OCE leaves. Similarly, OCE leaves maintained higher leaf Rubisco activities and protein than controls following dark chilling. Immunogold labelling studies with specific antibodies showed that Rubisco was present in Rubisco vesicular bodies (RVB) as well as in the chloroplasts of leaves from 8-week-old control and OCE plants. Western blot analysis of plants at 14 weeks after both genotypes had flowered revealed large increases in the amount of Rubisco protein in the OCE leaves compared to controls. These results demonstrate that CPs are involved in Rubisco turnover in leaves under optimal and stress conditions and that extra-plastidic RVB bodies are present even in young source leaves. Furthermore, these data form the basis for a new model of Rubisco protein turnover involving CPs and RVBs.

  18. Enzymological evidence for the function of a plastid-located pyruvate carboxylase in the Haptophyte alga Emiliania huxleyi: a novel pathway for the production of C4 compounds.

    Science.gov (United States)

    Tsuji, Yoshinori; Suzuki, Iwane; Shiraiwa, Yoshihiro

    2012-06-01

    Pyruvate carboxylase (PYC) catalyzes the β-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic β-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.

  19. Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat.

    Science.gov (United States)

    Zhang, HuiFang; Xu, WeiGang; Wang, HuiWei; Hu, Lin; Li, Yan; Qi, XueLi; Zhang, Lei; Li, ChunXin; Hua, Xia

    2014-09-01

    Using particle bombardment transformation, we introduced maize pepc cDNA encoding phosphoenolpyruvate carboxylase (PEPC) and ppdk cDNA encoding pyruvate orthophosphate dikinase (PPDK) into the C3 crop wheat to generate transgenic wheat lines carrying cDNA of pepc (PC lines), ppdk (PK lines) or both (PKC lines). The integration, transcription, and expression of the foreign genes were confirmed by Southern blot, Real-time quantitative reverse transcription PCR (Q-RT-PCR), and Western blot analysis. Q-RT-PCR results indicated that the average relative expression levels of pepc and ppdk in the PKC lines reached 10 and 4.6, respectively, compared to their expressions in untransformed plants (set to 1). The enzyme activities of PEPC and PPDK in the PKC lines were 4.3- and 2.1-fold higher, respectively, than in the untransformed control. The maximum daily net photosynthetic rates of the PKC, PC, and PK lines were enhanced by 26.4, 13.3, and 4.5%, respectively, whereas the diurnal accumulations of photosynthesis were 21.3, 13.9, and 6.9%, respectively, higher than in the control. The Fv/Fm of the transgenic plants decreased less than in the control under high temperature and high light conditions (2 weeks after anthesis), suggesting that the transgenic wheat transports more absorbed light energy into a photochemical reaction. The exogenous maize C4-specific pepc gene was more effective than ppdk at improving the photosynthetic performance and yield characteristics of transgenic wheat, while the two genes showed a synergistic effect when they were transformed into the same genetic background, because the PKC lines exhibited improved photosynthetic and physiological traits.

  20. Physiological investigation of C4-phosphoenolpyruvate-carboxylase-introduced rice line shows that sucrose metabolism is involved in the improved drought tolerance.

    Science.gov (United States)

    Zhang, Chen; Li, Xia; He, Yafei; Zhang, Jinfei; Yan, Ting; Liu, Xiaolong

    2017-06-01

    We compared the drought tolerance of wild-type (WT) and transgenic rice plants (PC) over-expressing the maize C 4 PEPC gene, which encodes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene, and evaluated the roles of saccharide and sugar-related enzymes in the drought response. Pot-grown seedlings were subjected to real drought conditions outdoors, and the yield components were compared between PC and untransformed wild-type (WT) plants. The stable yield from PC plants was associated with higher net photosynthetic rate under the real drought treatment. The physiological characters of WT and PC seedlings under a simulated drought treatment (25% (w/v) polyethylene glycol-6000 for 3 h; PEG 6000 treatment) were analyzed in detail for the early response of drought. The relative water content was higher in PC than in WT, and PEPC activity and the C 4 -PEPC transcript level in PC were elevated under the simulated drought conditions. The endogenous saccharide responses also differed between PC and WT under simulated drought stress. The higher sugar decomposition rate in PC than in WT under drought analog stress was related to the increased activities of sucrose phosphate synthase, sucrose synthase, acid invertase, and neutral invertase, increased transcript levels of VIN1, CIN1, NIN1, SUT2, SUT4, and SUT5, and increased activities of superoxide dismutase and peroxidase in the leaves. The greater antioxidant defense capacity of PC and its relationship with saccharide metabolism was one of the reasons for the improved drought tolerance. In conclusion, PEPC effectively alleviated oxidative damage and enhanced the drought tolerance in rice plants, which were more related to the increase of the endogenous saccharide decomposition. These findings show that components of C 4 photosynthesis can be used to increase the yield of rice under drought conditions. Copyright © 2017 Elsevier Masson SAS. All rights reserved.